Positron emission tomography radiotracers for fibroblast activation protein alpha

Abstract

Imaging and radiotherapeutic agents targeting fibroblast-activation protein-α (FAP-α) and their use in imaging and treating FAP-α related diseases and disorders are disclosed.

Description

POSITRON EMISSION TOMOGRAPHY RADIOTRACERS FOR FIBROBLAST ACTIVATION PROTEIN ALPHACROSS-REFERENCE TO RELATED APPLICATIONThis application claims the benefit of U. S. Provisional Application No. 63 / 747,681, filed January 21, 2025, which is incorporated by reference herein in its entirety.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under grants EB024495 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0002] Fibroblast activation protein, alpha (referred to herein as “FAP-a” or “FAP”) is a dimeric, 170-kDa cell surface protease that has exopeptidase and endopeptidase / gelatinase / collagenase activity. Brennen et al., 2012. FAP is abundantly expressed in more than 90% of reactive stromal fibroblasts of human epithelial cancers and has limited expression in normal, healthy tissues or adult tissues or fibroblasts of benign epithelial tumors. Kalluri, 2016; Garin-Chesa et al., 1990. It exists on the cell surface of cancer-associated fibroblasts (CAFs) and in a soluble, circulating form, Keane et al., 2014; Wu et al., 2021, CAFs are important in producing cytokines, chemokines, metabolites, enzymes, and extracellular matrix molecules that fuel malignancy. Kalluri, 2016. Sharma et al., 2022.

[0003] FAP, also called seprase. is an independent adverse prognostic factor for several malignancies, including colorectal, pancreatic, hepatocellular, and ovarian cancer. Fitzgerald and Weiner, 2020; Koerber et al., 2020; Mentlein et al., 2011; Hoppner et al., 2023. Several studies have shown that FAP-based imaging with positron emission tomography (PET) is a promising noninvasive and quantitative tool for the detection and therapeutic monitoring of cancer. Mona et al., 2022; Kratochwil et al., 2019.

[0004] Accordingly, FAP-targeted imaging agents have emerged as pan-cancer diagnostic tools while demonstrating improved diagnostic efficacy in primary and metastatic lesions compared with18F-FDG in numerous tumor types. Kratochwil et al., 2019; Chen et al., 2020; Kline et al., 2024. Furthermore, FAP-targeted PET imaging correlates with higher grade lesions, lymph node144429.601_P18646-02involvement, and lower overall survival for gastric cancer, glioma, ovarian cancer, and pancreatic ductal adenocarcinoma. Koerber et al., 2020; Pang et al., 2021; Röhrich et al., 2019; Windisch et al., 2020.

[0005] Several FAP targeting agents have been translated clinically for PET imaging. Among them, those employing68Ga-DOTA have garnered attention due to the ease of radiolabeling and the potential for easily switching to a therapeutic nuclide. Greifenstein et al., 2023; Loktev et al., 2019; Fendler et al., 2022; Baum et al., 2022; Kelly et al., 2021; Watabe et al., 2020. The development of18F-labeled compounds in high molar activity also would be desirable, however, because18F has favorable physical characteristics, including a high positron decay ratio (97%), relatively short half-life (109.7 min), low positron energy (maximum 0.635 MeV), and a short positron diffusion range (less than 2.4 mm), enabling images of higher resolution than those from68Ga. Emission properties of18F may further improve the detection of smaller lesions due to a lower partial volume effect. Sanchez-Crespo, 2013. The longer physical half-life of18F compared to68Ga (t1 / 2= 67.7 min) allows for facile transport to satellite imaging facilities that lack onsite cyclotron-based radionuclide production. Moreover, cyclotron-based production of18F is suggested to be more cost-effective than generator-based production of68Ga in the current clinical setting. Sahnoun et al., 2020.

[0006] The most extensively studied18F-labeled, FAP-targeted investigational agents include18F-AlF-FAPI-42 and18F-AlF-FAPI-74. Giesel et al., 2021; Lindner et al., 2021; Wang et al., 2021; Witek et al., 2024. In those compounds, the18F radionuclide is coordinated to an aluminum fluoride (Al-F coordinate bond) through a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator. Others have published similar analogs with modified linkers, for example,18F-A1F-P-FAPI and18F-A1F-FAPT. Hu et al., 2022; Huang et al., 2022.

[0007] A limited number of18F-labeled agents also have been developed in which the18F is covalently linked to the FAP targeting moiety using various non-metalated prosthetic groups, including tetrazine trans-cyclooctene ligation. Toms et al., 2020; Poulie et al., 2023.

[0008] Recently,18F covalently attached to small molecules was developed to accommodate one or two polar groups (either a methoxy substituent for the alcohol and vicinal diol groups or a quaternary ammonium group), providing improved pharmacokinetics. Tanc et al., 2024.

[0009] Yang et al. developed an FAPI-targeted theranostic agent containing the 4-quinolinoyl-glycyl-2-cyanopyrrolidine pharmacophore and an SiFA prosthetic group with a covalent18F and244429.601_P18646-02DOTAGA moiety in a single molecule. Yang et al., 2023. That agent proved capable of imaging FAP-expressing tumor in relevant experimental models, but the uptake in other organs was high, perhaps due to the highly hydrophobic nature of SiFA. Despite tumor-specific uptake, most published compounds do not perform as well as the leading chelator-based agents, such as18F-A1F-FAPI-74.SUMMARY

[0010] In some aspects, the presently disclosed subject matter provides a compound of formula (I):xI

[0011] C-La-A(I);

[0012] wherein:

[0013] A is a targeting moiety for fibroblast activation protein alpha (FAP-a);

[0014] C is a chelating group comprising a naturally occurring non-radioactive isotope of a metal;

[0015] P is a prosthetic group;

[0016] X is a radioisotope of a halogen;

[0017] Lais a tri-functionalized linker capable of forming a chemical bond with A, Lb, and C;

[0018] Lb is a bi-functionalized linker capable of forming a chemical bond with Laand P; and stereoisomers and pharmaceutically acceptable salts thereof.

[0019] In particular aspects, the FAP-alpha binding moiety comprises:

[0020]

[0021] In particular aspects, P-X comprises:

[0023] wherein X is selected from18F,124I,1251,131I, and211At.

[0024] In particular aspects, P-X is:344429.601_P18646-02L JJ H — I *

[0025] 18F N

[0026] In particular aspects, the linker, La, comprises:

[0027] 6 orN'— °y

[0028] In particular aspects, the linker. Lb, comprises a Ci-Cs alkylene chain.

[0029] In particular aspects, the chelator comprises 1,4, 7, 10-tetraazacyclododecane- 1,4, 7 -tris-acetic acid-10 monoamide (DOTA-monoamide):HOOC HOOC N NN—

[0030] HOOC-J°.

[0031] In certain aspects, the naturally occurring non-radioactive isotope of a metal is selected from Cu, Pb, Ac, Lu, Ga, Tb, Y, In, Re, Sm, Zr, Bi, Sc, Ho, Ra, Th, and Al. In particular aspects, the naturally occurring non-radioactive isotope of a metal is selected from27Al,45Sc,63Cu.65Cu,69Ga,71Ga,89Y,90Zr,91Zr,92Zr,94Zr,96Zr,113In,115In,144Sm,147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th.

[0032] In particular aspects, the compound of formula (I) comprises:

[0034] wherein M is selected from113In / 115In,45Sc, and69Ga / 71Ga.

[0035] In more particular aspects, the compound of formula (I) comprises:18F

[0036] o

[0037] wherein M is69Ga / 71Ga.444429.601_P18646-02

[0038] In some aspects, the presently disclosed subject matter provides a pharmaceutical composition comprising the compound of formula (I). In certain aspects, the formulation comprises one or more of pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

[0039] In other aspects, the presently disclosed subject matter provides a method for imaging a disease or disorder associated with fibroblast- activation protein-a (FAP-a), the method comprising administering a compound of formula (I), or a pharmaceutical composition thereof, and obtaining an image.

[0040] In other aspects, the presently disclosed subject matter provides a method for inhibiting fibroblast-activation protein-a (FAP-a), the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutical composition thereof.

[0041] In certain aspects, the (FAP-a)-related disease or disorder is selected from a proliferative disease, a disease characterized by tissue remodeling and / or chronic inflammation, a disorder involving endocrinological dysfunction, and a blood clotting disorder.

[0042] In particular aspects, the proliferative disease is selected from breast cancer, colorectal cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, melanoma, fibrosarcoma, bone and connective tissue sarcomas, renal cell carcinoma, giant cell carcinoma, squamous cell carcinoma, gastric cancer, a glioma, and adenocarcinoma, including pancreatic ductal adenocarcinoma.

[0043] In other aspects, the presently disclosed subject matter provides a method for synthesizing:18F

[0045] the method comprising:

[0046] (a) providing a trimethylammonium precursor compound (3):544429.601_P18646-02

[0048] (b) contacting the trialkylammonium precursor compound (3) with18F / 4,7, 13, 16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane in the presence of a base in a polar, aprotic solvent at a first tempearture to form [18F]1:18F

[0050] (c) contacting ([18F]1) with M3+in a buffer at a second temperature to form:wherein M is a naturally occurring non-radioactive isotope or a mixture of isotopes of a metal. As provided herein below, a naturally occurring metal isotope is designated asnatM.

[0052] In certain aspects, M is selected fromnatCu,natPb,natAc,natLu,natGa,natTb,natY,natIn,natRe,natSm,natZr,natBi,natSc,natHo.natRa,natTh, andnatAl. In particular aspects, M is selected from27Al,45Sc,63Cu,65Cu,69Ga,71Ga,89Y,90Zr,91Zr,92Zr,94Zr,96Zr,113In,115In,144Sm,147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th. In more particular aspects, M is selected fromnatIn,natSc, and “atGa. In certain aspects, M3+is a salt selected fromnatInCl3,natScCl3,natGaCl3,natIn (NO3)3,natSc(NO3)3, andnatGa (NO3)3.

[0053] In certain aspects, [18F]-natM-l has a radiochemical yield of about 25%. In certain aspects,[18F]-natM-l has a radiochemical purity greater than about 95%. In certain aspects, [18F]-natM-l has a molar (specific) radioactivity between about 2000 to about 5000 Ci / mmol.

[0054] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.644429.601_P18646-02BRIEF DESCRIPTION OF THE FIGURES

[0055] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0056] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0057] FIG. 1 shows the chemical structures of FAP-based agents in clinical and pre-clinical development, including18F-FAPI-42,18F-FAPI-74,18F-A1F-P-FAPI,18F-A1F-FAPT,18F-10,18F-FGlc-FAPI.

[0058] FIG. 2A, FIG. 2B, and FIG. 2C demonstrates that compound 1 is a high-affinity hFAP-a inhibitor comparable to FAPI-04. (FIG. 2A) Chemical structure of QCP-2, 1 and FAPI-04; (FIG.2B-FIG. 2C) Surface representation of the active site of hFAP-a (PDB ID: 1Z68) with (FIG. 2B) 1 (cyan colored stick) and (FIG. 2C) FAPI-04 (yellow colored stick). The protein is depicted as solid gray surface and residues in SI and S2 pockets are colored green and deep salmon respectively. A few residues in the hydrophobic pocket SI (Tyr625, Tyr656) and S2 (Trp623, Tyr541, Pro544, Tyr625, Phe350 and Phe351) surrounding the catalytic triad residue (Ser624) are shown and indicated by a 1 -letter code. While 1 and FAPI-04 both occupy the catalytic binding pocket of hFAP-a, their binding modes differ significantly. The pyrrolidine moiety of 1 is buried in the SI hydrophobic pocket close to S624, whereas that of FAPI-04 is situated in the S2 hydrophobic pocket with piperazine moiety near the S 1 pocket.

[0059] FIG. 3 A, FIG. 3B, FIG. 3C, and FIG. 3D shows the structural modification of FAPI-04 and QCP-2 to accommodate 6-fluoronicotinic acid prosthetic group creates the18F-FPyQCP compounds for18F-labeling. (FIG. 3A) Chemical structure of111In-1 and68Ga-l. (FIG. 3B) In vivo biodistribution of111In-1 in selected organs from NOD / SCID mice (n = 4) bearing U87 xenografts. (FIG. 3C-FIG. 3D) PET / CT imaging of (FIG. 3C)68Ga-l (approximately 74 MBq) in FAP(+) U87 tumor bearing male NOD / SCID mice during 30-60 min (n = 2), (FIG. 3D) In vivo biodistribution of68Ga-l in selected organs from NOD / SCID mice (n = 3 or 4) bearing U87 xenografts.

[0060] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F show structures and tissue biodistribution data18F-1,18F-natIn-l and18F-FPyQCP (18F-natGa-l). (FIG. 4A) Chemical structures of18F-1,18F-natIn-l and18F-FPyQCP. (FIG. 4B) Tissue biodistribution data for18F-1744429.601_P18646-02without and with blocker at 1 h post-injection in selected organs from mice (n = 3 or 4) bearing U87 xenograft. Male NOD / SCID mice with human tumor xenografts were injected with 0.74 MBq at 1 hour post injection. (FIG. 4C-FIG. 4E) Tissue biodistribution data of18F-natIn-l (FIG. 4D) 18and F-FPyQCP (FIG. 4E) without and with blocker at 1 h post-injection in selected organs from male NOD / SCID mice (n = 4) bearing U87 xenografts. Blocking-dose mice received FAPI-04 (30 nmol) 5 min prior to radiotracer injection. (FIG. 4C- FIG. 4D) Head-to-head comparison of (FIG.4E) biodistribution and (FIG. 4F) tumor-to-organ ratios of18F-1,18F-natIn-l and18F-FPyQCP in selected tissues harvested from male NOD / SCID mice bearing U87 xenografts. Error bars indicate SD (n=4). T=tumor; B=blood; S=salivary gland; L= liver; M=muscle; Bn=bone. Statistical significance was established using Two-way ANOVA followed by a Sidak’s multiple comparison test (ns = not significant; * P< 0.05 ** P < 0.01; *** P < 0.001).

[0061] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H, and FIG. 51 show a head-to-head comparison in FAP+ human tumors showing improved performance of18F-FPyQCP compared to18F-AlF-FAPI-74. (FIG. 5A) FAP surface expression in HT-1080, HT-1080-FAP and Capan-2 and U87 by flow cytometry; (FIG. 5B) Geometric mean of FAP expression normalized to unstained in HT-1080, HT-1080-FAP and Capan-2 and U87 (flow cytometry); (FIG.5C- FIG. 5E) Biodistribution at 1 h after injection (n = 5 / time) without and with blocker (10 nmol / mouse) in mice bearing (FIG. 5C) U87, (FIG. 5D) Capan-2, and (FIG. 5E) HT-1080-FAP xenografts. Mice were injected with 0.74 MBq in 150 pL saline. Blocking-dose mice received FAPI-04 (30 nmol) 5 min prior to radiotracer injection (n = 5). (FIG. 5F) H& E staining of whole tumors (top panel, scale bar, 10 mm (HT-1080-FAP) and 5 mm (Capan-2 and U87). Representative images (scale bar, 100 pM; x20) of H& E (middle panel) and FAP (bottom panel) staining were indicated by a, b and c in HT-1080-FAP, Capan-2 and U87, respectively. (FIG. 5G) QuPath quantification of FAP-positive cells in the tumors. (FIG. 5H) Tumor-to-organ ratios of18F-FPyQCP and18F-AlF-FAPI-74 in selected organs. Error bars indicate SD (n = 5). Two-way ANOVA followed by a Sidak’s multiple comparison test (ns = not significant; * P < 0.05 ** P < 0.01; *** P< 0.001). FIG. 51 is a comparison of uptake of radiotracers [18F]1, [18F]-natIn-l, [18F]-natSc-l and [18F] -natGa -1 (18F-FPyQCP) in U87 xenografts in NOD / SCID mice in baseline and blocking experiments.

[0062] FIG. 6 A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG. 6E show whole-body decay corrected PET imaging of18F-FPyQCP and18F-AlF-FAP-74 in human gliblastoma U87 tumor xenografts bearing 844429.601_P18646-02male NOD / SCID mice. FIG. 6A- FIG. 6B. Dynamic PET and PET / MR imaging of18F-FPyQCP during 0-90 minutes post-injection of 7.4 ± 0.2 MBq dose and time-activity curves (TAC) of tumor and kidneys. White arrows indicate the location of the tumors. FIG. 6C. Static decay-corrected whole-body coronal PET images (prone and supine) of18F-FPyQCP and18F-AlF-FAP-74 at indicated time-points after intravenous radiotracer administration via tail-vein injection. Red arrows indicate the location of the tumors. FIG. 6D. Static decay-corrected whole-body coronal PET / MR images of a pair mice with (left) and without blockade (right). Blocking dose, 30 nmol FAPI-06 per mouse, co-injection.18F-FPyQCP. (Top) and18F-AlF-FAP-74 (Bottom). Mice were injected with 7.4 ± 0.2 MBq within 5 min and imaging was done at 1 h after injection. FIG 6E shows comparison of tumor uptake (SUV) after injection of18F-FPyQCP and18F-AlF-FAP-74.

[0063] FIG. 7A and FIG. 7B show (FIG. 7A) whole body PET imaging of18F-FPyQCP (212.7 MBq, 5.7 mCi) and18F-FAPI-74 (246.4 MBq, 6.6 mCi) in the same non-human primate (Papio anubus) at 90 min post-injection. Red arrowheads depict the gallbladder (FIG. 7B) Cross-sectional PET / CT at the level of the gallbladder (red arrowheads). Note faint uptake within pancreas for18F-FPyQCP (yellow dotted line). K = kidney; GI = gastrointestinal tract.

[0064] FIG. 8A, FIG. 8B, and FIG. 8C show the radiochemical synthesis of111In-l,68Ga-l,18F-1,18F-natIn-l, and18F-FPyQCP (18F-natGa-l). (FIG. 8A) Schematic diagram of radiolabeling of the radiometal labeled agents,111In-1 and68Ga-1. (FIG. 8B) The radiolabeling scheme for the18F-labeled agents,18F-1,18F-natIn-l, and18F-FPyQCP (18F-natGa-l). The18F-labeled agents were synthesized using semi- automated radiochemistry module. (FIG. 8C) HPLC chromatogram of the preparative reaction mixture of18F-FPyQCP (left) and the purified tracer (right).

[0065] FIG. 9A and FIG. 9B are preparative HPLC chromatograms of111In-1; FIG. 9A) peak at X = 220 nm, FIG. 9B) radioactive peak. UV peak at 16.2 min is related to the unbound ligand 1.

[0066] FIG. 10A and FIG. 10B are preparative HPLC chromatograms of68Ga-l; FIG. 10A) peak at X = 220 nm, FIG. 10B) radioactive peak. UV peak at 12.2 min is related to the unbound ligand 1.

[0067] FIG. 11 A and 1 IB are a cartoon representation of human FAP (PDB ID 1Z68) with 1. The docked ligands and bound 1 are shown in a stick model, and the active site residues (S624, D702 and H734) are indicated by a 1-letter code. The hydrogen, and hydrophobic interactions are drawn as red and blue dotted lines respectively and the lengths are indicated. Image was created using PyMol version 2.5.5.944429.601_P18646-02

[0068] FIG. 11C is a 2D LigPlot representation of the docking interaction of human FAP (PDB ID 1Z68) with 1. Receptor residues involved in hydrophobic interactions are represented by brick red spoked arcs, and hydrogen bonding are colored green. The hydrogen-bonding interactions are shown by green dotted lines.

[0069] FIG. 12A and FIG. 12B are cartoon representations of human FAP (PDB ID 1Z68) with FAPI-04. The docked ligands and bound FAPI-04 are shown in a stick model, and the active site residues (S624, D702 and H734) are indicated by a 1 -letter code. The hydrogen, and hydrophobic interactions are drawn as red and blue dotted lines respectively and the lengths are indicated. Image was created using PyMol version 2.5.5.

[0070] FIG. 12C is a 2D LigPlot representation of the docking interaction of human FAP (PDB ID 1Z68) with FAPI-04. Receptor residues involved in hydrophobic interactions are represented by brick red spoked arcs, and hydrogen bonding are colored green. The hydrogen-bonding interactions are shown by green dotted lines.

[0071] FIG. 13 is preparative HPLC chromatograms of18F-1. Top. radioactive peak (red) and bottom, UV peak at 6 min is related to the unbound ligand 1.

[0072] FIG. 14 shows spike HPLC chromatograms of18F-1 and UV peak at 6 min is related to the unbound ligand 1.

[0073] FIG. 15A and FIG. 15B are quality control HPLC chromatograms of18F-natIn-l; FIG.15A) peak at 13.3 min is related to18F-natIn-l, FIG. 15B) UV peak at 6 min is related to the unbound 1.

[0074] FIG. 16 is a QC HPLC chromatograms of18F-natIn-l. Top, radioactive peak at 4.3 min; bottom, associated UV peak at 4.3 min.

[0075] FIG. 17 is a preparative HPLC chromatograms of18F‘A1F-FAPL74; radioactive peak (top) at 5.10 min is related to peak8F’AlF-FAPI-74, and UV peak at = 220 nm at 4.2 min (bottom) is related to the unbound FAPI-74.

[0076] FIG. 18 is a stability study. The radiotracer was synthesized as described herein and formulated in 10 mL of 10% ethanol in saline (which contained 67.7 mg sodium L-Ascorbate). The mixture was then left at room temperature for 2 hours before further QC HPLC analysis, when a 10 pL sample was taken from the mixture.1044429.601_P18646-02

[0077] FIG. 19A, FIG. 19B, and FIG. 19C show a flow cytometry analysis confirming high cell surface expression of FAP in HT-1080-FAP (FIG. 19A), and U87 cells (FIG. 19C) and low expression in Capan-2 (FIG. 19B) and HT-1080 cell line (FIG. 19A).

[0078] FIG. 20 is a decay-corrected PET / MR imaging of 18F-FPyQCP in a mouse bearing U87 tumor (n=2), confirming high tumor uptake and retention tumor up to 4 h post-injection. This mouse was subjected to a dynamic scan after 150 min post-injection.

[0079] FIG. 21 is an1H-NMR Spectrum (500 MHz, DMSO-6) of compound .

[0080] FIG. 22 is an1H-NMR Spectrum (500 MHz, DMSO-6) of compound .

[0081] FIG. 23 is an1H-NMR Spectrum (500 MHz, DMSO-6) of .

[0082] FIG. 24 is an1H-NMR Spectrum (500 MHz, DMSO-6) of .

[0083] FIG. 25 is an1H-NMR Spectrum (500 MHz, DMSO-6) of , .

[0084] FIG. 26 is an HRMS report of 1.

[0085] FIG. 27 is an HRMS report of D-l.

[0086] FIG. 28 is an HRMS Report ofnatGa-l.

[0087] FIG. 29 is an HRMS Report ofnatIn-l.DETAILED DESCRIPTION

[0088] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0089] Fibroblast-activation protein-a (FAP-a) is a type II integral membrane serine protease of the prolyl oligopeptidase family, which are distinguished by their ability to cleave the Pro-AA peptide bond (where AA represents any amino acid). FAP-a exists as a homodimer to carry out its enzymatic function. It has been shown to play a role in cancer by modifying bioactive signaling peptides through this enzymatic activity (Kelly, et al.. 2005; Edosada, et al., 2006). FAP-a expression has been detected on the surface of fibroblasts in the stroma surrounding greater than1144429.601_P18646-0290% of the epithelial cancers, including, but not limited to, malignant breast, colorectal, skin, prostate, pancreatic cancers, and the like, and inflammation diseases, including, but not limited to, arthritis, fibrosis, and the like, with nearly no expression in healthy tissues. Inhibitors selectively targeting FAP-a has been reported (Lo, et al., 2009; Tsai, et al., 2010; Ryabtsova, et al., 2012; Poplawski, et al., 2013; Jansen, et al., 2013; Jansen, et al., 2014).

[0090] More particularly, FAP-a expression has been detected on the surface of fibroblasts in the stroma surrounding >90% of the epithelial cancers examined, including malignant breast, colorectal, skin, prostate, and pancreatic cancers. (Garin-Chesa, et al., 1990; Rettig, et al., 1993; Tuxhorn, et al., 2002; Scanlan, et al., 1994). It is a characteristic marker for carcinoma-associated-fibroblast (CAF), which plays a critical role in promoting angiogenesis, proliferation, invasion, and inhibition of tumor cell death. (Allinen, et al., 2004; Franco, et al., 2010). In healthy adult tissues, FAP-a expression is only limited to areas of tissue remodeling or wound healing. (Scanlan, et al., 1994; Yu, et al., 2010; Bae, et al., 2008; Kraman, et al., 2010). In addition, FAP-a-positive cells are observed during embryogenesis in areas of chronic inflammation, arthritis, and fibrosis, as well as in soft tissue and bone sarcomas. (Scanlan, et al., 1994; Yu, et al., 2010). These characteristics make FAP-a a potential imaging and radiotherapeutic target for cancer and inflammation diseases.

[0091] Because FAP-a is expressed in tumor stroma, anti-FAP antibodies have been investigated for radioimmunotargeting of malignancies, including murine F19. sibrotuzumab (a humanized version of the F19 antibody), ESC11, ESC14, and others. (Welt, et al., 1994; Scott, et al., 2003; Fischer, et al., 2012). Antibodies also demonstrated the feasibility of imaging inflammation, such as rheumatoid arthritis. (Laverman, et al., 2015). The use of antibodies as molecular imaging agents, however, suffers from pharmacokinetic limitations, including slow blood and non-target tissue clearance (normally 2-5 days or longer) and non-specific organ uptake. Low molecular weight (LMW) agents demonstrate faster pharmacokinetics and a higher specific signal within clinically convenient times after administration. They also can be synthesized in radiolabeled form more easily and may offer a shorter path to regulatory approval. (Coenen, et al., 2010; Coenen, et al., 2012; Reilly, et al., 2015).

[0092] The presently disclosed subject matter provides, in part, compound comprising a FAP-a selective targeting moiety that can be modified with a metal chelating complex and a radiolabeled prosthetic group, thus providing a platform for the imaging and radiotherapy targeting FAP-a.1244429.601_P18646-02

[0093] To overcome issues with current FAP-based PET imaging agents and considering the increased importance of FAP-based PET imaging in oncology, the presently disclosed subject matter provides compounds that employ a 6-fluoronicotinamide moiety to introduce18F. That prosthetic group has been leveraged to produce18F-labeled compounds in high yield and molar activity in an industrial scale preparation in a single step using readily available intermediates. Ravert et al., 2017; Rong et al.. 2023.

[0094] Additionally, in vivo metabolic stability of such labeled analogs has been well-documented in patient studies. Unlike the hydrophobic prosthetic groups fluorobenzamide or SiFA, the 6-fluoronicotinamide moiety is hydrophilic and does not promote high non-specific binding. Notably, an approved prostate-specific membrane antigen (PSMA)-based PET imaging agent,18F-DCFPyL (piflufolastat F18), utilizes the same prosthetic group. Szabo et al., 2015.

[0095] To this end, International PCT Patent Application Publication No. WO2022 / 212958 Al, for Heterobivalent and Homobivalent Agents Targeting Fibroblast Activation Protein Alpha and / or Prostate-Specific Membrane Antigen, to Ray et al., published October 6, 2022 (hereinafter WO2022 / 212958), which is incorporated by reference in its entirety, describes a series of radiopharmaceutical agents targeting FAP.

[0096] Certain compounds disclosed in WO2022 / 212958 include a 6-fluoronicotinamide prosthetic group, a chelator and a FAP-binding moiety connected with linkers. The radiotracers were radiolabeled with either18F or a radiometal attached to DOTA or another chelator. Several radiolabeled compounds disclosed in WO2022 / 212958, including111In-SRI-08-15 demonstrated interesting imaging properties in U87 tumors (see WO 2022 / 212958 Al, page 143, for mouse data). The chemical structures of chelator SRI-08-15 and radiolabeled [111In]SRI-08-15, are shown immediately herein below:

[0097] In some embodiments, the presently disclosed subject matter provides chelates of18F-radiolabeled SRI-08-15 ([18F]), and related compounds of formula (I), with various non-1344429.601_P18646-02radioactive natural metal (natM) cations and their PET imaging properties. The presently disclosed subject matter, in some embodiments, demonstrates that chelation with a “cold” radiometal, e.g., a non-radioactive natural metal (natM), improves imaging properties and tumor uptake of the radiotracers.

[0098] Accordingly, in some embodiments, the presently disclosed subject matter provides potent and selective low-molecular- weight (LMW) ligands of FAP-a, i.e.. an FAP-a selective inhibitor, conjugated with a prosthetic radiolabeling group, and metal chelators, which enable in vivo nuclear imaging (e.g., PET and SPECT) and radiotherapy targeting FAP-a.

[0099] The presently disclosed compounds of formula (I) are small molecules and / or low molecular weight compounds. As used herein, the terms “small molecule” and “low molecular weight” are used interchangeably and refer to a compound having a molecular weight between about 50 Dalton (Da) to about 1,500 Da, including 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, and 1500 Da.

[0100] The term of “low molecular weight” is well accepted in the chemical arts, and its meaning is clear to one of ordinary skill in the art. In particular, when used in the scientific references in the chemical arts, one of ordinary skill in the art would recognize that low molecular weight compounds would have a molecular weight of typically from about 50 Daltons to about 1,500 Daltons. See e.g., K Beebe et al., Clin Transl Sci. 2014 Feb; 7(1): 74-81 (“Metabolomics is often described as a systematic study of the low molecular weight (approximately 50-1,500 Da) metabolites chemicals) within a given sample”); A. Ferreira et al., J. Agric. Food Chem. 2014, 62, 6784-6793 (“Metabolites are a group of low molecular weight substances (50-1500 Da) that includes amino acids, fatty acids...”); C. Llewellyn et al.. Progress in Oceanography, Volume 137, p. 421-433 (Metabolomics involves the non-targeted unbiased analysis of large suites of low molecular weight organic molecules or metabolites (typically 50-1500 Da)...); see also https: / / www.ebi.ac.uk / training / online / courses / metabolomics-introduction / what-is / small-molecules / (“A small molecule (or metabolite) is a low molecular weight organic compound, typically involved in a biological process as a substrate or product. Metabolomics usually studies small molecules within a mass range of 50 - 1500 daltons (Da)”).

[0101] Importantly, the presently disclosed compounds can be modified, e.g., conjugated with, labeling groups without significantly losing their potency. The presently disclosed approach allows for the convenient labeling of the FAP-a ligand with PET or SPECT isotopes. Further, the1444429.601_P18646-02presently disclosed approach allows for the radiolabeling of the FAP-a targeting compound with radiotherapeutic isotopes FAP-a related radiotherapy.

[0102] Compounds o f Formula (I)

[0103] In some embodiments, the presently disclosed subject matter provides a compound of formula (I):xb / I

[00104] C“La— A(I);

[0105] wherein:

[0106] A is a targeting moiety for fibroblast activation protein alpha (FAP-a);

[0107] C is a chelating group comprising a naturally occurring non-radioactive isotope of a metal;

[0108] P is a prosthetic group;

[0109] X is a radioisotope of a halogen;

[0110] Lais a tri-functionalized linker capable of forming a chemical bond with A, Lb, and C;

[0111] Lb is a bi-functionalized linker capable of forming a chemical bond with Laand P;

[0112] and stereoisomers and pharmaceutically acceptable salts thereof.

[0113] Naturally occurring, non-radioactive isotopes are designated herein asnatM.Representative naturally occurring, non-radioactive isotopes include, but are not limited to,27A1,45Sc,63Cu,65Cu,69Ga,71Ga,89Y,90Zr,91Zr,92Zr,94Zr,96Zr,113In,115In,144Sm,147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th. In particular embodiments, the naturally occurring, non-radioactive isotope is selected from45Sc,69Ga / 71Ga, and113In / 115In. In more particular embodiments, the naturally occurring, nonradioactive isotope is69Ga / 71Ga. One of ordinary skill in the art would recognize that naturally occurring, non-radioactive isotopes are substantially free from radioactive isotopes. As used herein, the term “substantially free” refers to naturally occurring isotopes having, in some embodiments, less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, and 0.0% of a radioactive isotope.

[0114] Suitable FAP-a specific targeting moieties are provided in International PCT Patent Application Publication No. WO2019 / 083990 A2 to Yang et al., for Imaging and Radiotherapeutic Agents Targeting Fibroblast- Activation Protein-a (FAP-a), published May 2, 2019, which is incorporated herein by reference in its entirety.1544429.601_P18646-02

[0115] In certain embodiments, A comprises an FAP-a targeting moiety having the following structure:^5xR?x

[0116] R6X (A’);

[0117] wherein each y is independently an integer selected from 0, 1, and 2;

[0118] Rix, R2X, and Rsx', are each independently selected from H, OH, halogen, Ci-ealkyl, -O-Ci- ealkyl, and -S-Ci-6alkyl;

[0119] R3X is selected from H, -CN, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C- S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and 5-tetrazolyl;

[0120] R4X is selected from H, straight-chain or branched C1-6 alkyl, -(CH2)q4-aryl, and hydroxylsubstituted straight-chain or branched C1-6 alkyl, wherein q4 is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

[0121] Rs. Rex, and R?x are each independently selected from H, -OH, oxo, halogen, -Ci-ealkyl, - O-Ci ealkyl, -S-Ci ealkyl, -NRgxR x, -ORi2x, -Het2 and -Ar2; each of Ci ealkyl being optionally substituted with from 1 to 3 substituents selected from -OH and halogen;

[0122] Rsx. R9, and Ri2x are each independently selected from H, -OH, halo, -Ci-6alkyl, -O-Ci- ealkyl, -S-Ci-ealkyl, and -An;

[0123] Riox, Riix, Ri3x and Ri4 are each independently selected from H, -OH, halogen, -Ci-6alkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl; An, An and An are each independently a 5- or 6-membered aromatic monocycle optionally comprising 1 or 2 heteroatoms selected from O, N and S; each of An, An and An being optionally and independently substituted with from 1 to 3 substituents selected from -NRioxRiix, -Ci-ealkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl;

[0124] Het2 is a 5- or 6-membered non-aromatic monocycle optionally comprising 1 or 2 heteroatoms selected from O, N and S; Het2 being optionally substituted with from 1 to 3 substituents selected from -NRi3xRi4x, -Ci-ealkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl; / - \

[00125] v is 0, 1, 2, or 3; and1644429.601_P18646-02

[0126] represents a 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S;

[0127] whereinindicates a point of attachment of the FAP-a binding ligand to the linker, La, wherein the point of attachment can be through any of the carbon atoms of the 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle thereof;

[0128] and stereoisomers and pharmaceutically acceptable salts thereof.

[0129] In particular embodiments, R4x is selected from H, -CH3, -CFF-phenyl, -CH(CH3)2, -CH2-OH, and -CH(OH)CH3. / - \

[0130] In certain embodiments,is selected from:

[0131]

[0132] wherein * indicates the point of attachment of the 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle to -(CH2)V-

[0133] In certain embodiments, A comprises an FAP-a targeting moiety having the following structure:1744429.601_P18646-02

[0135] wherein indicates a point of attachment of the F AP-a binding ligand to the linker, L3, wherein the point of attachment can be through any of carbon atoms 5, 6, 7, or 8 of the quinolinyl ring thereof; and stereoisomers and pharmaceutically acceptable salts thereof.

[0136] In certain embodiments, A is selected from:

[0138] In certain embodiments, A is selected from:

[0140] and stereoisomers thereof.

[0141] In certain embodiments, A is selected from:1844429.601_P18646-02

[0143] Suitable FAP inhibitors are disclosed in International PCT Patent Application No. WO2019 / 154886 for FAP Inhibitor, to Haberkorn et al., published August 15, 2019, which is incorporated herein by reference in its entirety. Representative FAP ligands, linkers, and reporting moieties include compounds of formula (I):

[0144]

[0145] wherein:

[0146] Q, R, U, V, W, Y, Z are individually present or absent under the proviso that at least three of Q, R. U, V, W, Y, Z are present;

[0147] Q, R, U, V, W, Y, Z are independently selected form the group consisting of O, CH2, NR4, C=O, C=S, C=NR4, HCR4and R4CR4, with the proviso that two oxygen atoms are not directly adjacent to each other;

[0148] R1and R2are independently selected from the group consisting of -H, -OH, halo, C1-6-alkyl, -O-Ci-6-alkyl, -S-Ci-6-alkyl;

[0149] R3is selected from the group consisting of -H, -CN, -B(OH)2, -C(O) -alkyl, -C(O)-aryl-, -C=C-C(O)-aryl, -C=C-S(O)2-aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and 5-tetrazolyl;

[0150] R4is selected from the group consisting of -H, -Ci-6-alkyl, -O-Ci-6-alkyl, -S-Ci-6-alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, and -Ci-6-aralkyl, each of said -Ci-6-alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z;

[0151] R5is selected from the group consisting of -H, halo, and Ci-6-alkyl;

[0152] R6, and R7are independently selected from the group consisting of -H,g

[0153] , under the proviso that R6and R7are not at the same time H, wherein L is a linker, wherein D, A, E, and B are individually present or absent, preferably wherein at least A, E, and B are present, wherein when present:

[0154] D is a linker;

[0155] A is selected from the group consisting of NR4, O, S. and CH2;

[0156] E is selected from the group consisting of Ci-ealkyl,1944429.601_P18646-02■01;FF F F

[0157]

[0158] wherein i is 1, 2, or 3;

[0159] wherein j is 1, 2, or 3;

[0160] wherein k is 1, 2, or 3;

[0161] wherein m is 1, 2, or 3;

[0162] B is selected from the group consisting of S, NR4, NR4-O, NR4-Ci-6-alkyl, NR4-CI-6- alkyl- NR4, and a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein NR4-Ci-6-alkyl-NR4and the N- containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of Ci-6- alkyl, aryl, Ci-6-aralkyl; and

[0163] R8is selected from the group consisting of radioactive moiety, chelating agent, fluorescent dye, a contrast agent, and combinations thereof;

[0164] is a 1-naphthyl moiety or a 5 to 10- membered N-containing aromatic or non- aromatic mono- or bicyclic heterocycle, wherein there are 2 ring atoms between the N atom and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom;

[0165] or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof.

[0166] In certain embodiments, A is an FAP-a targeting moiety or ligand having the structure of:

[0167] N;

[0168] wherein Xi and X2 are each independently H or F.2044429.601_P18646-02

[0169] In particular embodiments, the FAP inhibitor disclosed in WO2019 / 154886 is a compound, including the FAP ligand, linker, and reporting moiety, disclosed in one or more of Table 1, Table 2, Table 3, Table 4, and Table 5, or any compound disclosed on page 44, line 1, through page 75, line 6, which is incorporated herein by reference, including, but not limited to FAPI-1, FAPI-2, FAPI-3, FAPI-4, FAPI-5, FAPI-6, FAPI-7, FAPI-8, FAPI-9, FAPI-10, FAPI-11, FAPI-12, FAPI-13, FAPI-14, FAPI-15. FAPI-16, FAPI-17, FAPI-18, FAPI-19. FAPI-20. FAPI-21, FAPI-22, FAPI-23, FAPI-24, FAPI-25, FAPI-26, FAPI-27, FAPI-28, FAPI-29, FAPI-30, FAPI-31, FAPI-32, FAPI-33, FAPI-34, FAPI-35, FAPI-36, FAPI-37, FAPI-38, FAPI-39, FAPI-40, FAPI-41, FAPI-42, FAPI-43, FAPI-44, FAPI-45, FAPI-46, FAPI-47, FAPI-48, FAPI-49, FAPI-50, FAPI-51, FAPI-52, FAPI-53, FAPI-54, FAPI-55, FAPI-56, FAPI-57, FAPI-58, FAPI-60, FAPI-61, FAPI-62, FAPI-63, FAPI-64. FAPI- FAPI-65, FAPI-66. FAPI-67, FAPI-68, FAPI-69, FAPI-70, FAPI-71, FAPI-72, FAPI-73, FAPI-74, FAPI-75, FAPI-76, FAPI-77, FAPI-78, and FAPI-79, each of which incorporated herein by reference.

[0170] In some embodiments, the FAP-a ligand includes a substituted (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine scaffold disclosed in Jansen et al., Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold. ACS Med Chem Lett.2013 Mar 18;4(5):491-6; Jansen et al., Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP). J Med Chem. 2014 Apr 10;57(7):3053-74, each of which is incorporated by reference in their entirety. Such FAP-a ligands include the following structure:N

[00171]

[0172] wherein:

[0173] Xi and X2 are each independently H or F; and

[0174] R15x is selected from the group consisting of H, C1-6 alkyl, halogen, trihalomethoxyl, C1-6 alkoxyl, and 4-methoxyphenyl.

[0175] Also included are FAP ligands disclosed in Roy et al., Design and validation of fibroblast activation protein alpha targeted imaging and therapeutic agents, Theranostics 2020, 10 (13), 5778-5789, which is incorporated herein by reference in its entirety, including, but not limited to:2144429.601_P18646-02

[00176] N

[0177] In certain embodiments, A comprises a targeting moiety for FAP-a having the following

[0179] In certain embodiments, A comprises an FAP-a targeting moiety having the following structure:

[0181] wherein:

[0182] each y is independently an integer selected from 0, 1, and 2;

[0183] R1x, R2x, and R3x', are each independently selected from H, OH, halogen, Ci-ealkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl;

[0184] whereinindicates a point of attachment of the FAP-a binding ligand to the linker, L3, wherein the point of attachment can be through any of the carbon atoms of the 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle thereof;

[0185] and stereoisomers and pharmaceutically acceptable salts thereof.

[0186] In certain embodiments, P-X is selected from:2244429.601_P18646-02X

[0188] wherein:

[0189] each X is independently a radioisotope of a halogen;

[0190] each n is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, and 20;

[0191] each carbon of thenalkylene chain can be substituted with C1-C4 alkyl; and

[0192] each Ryand Ry’ is independently H or C1-C4 alkyl.

[0193] In certain embodiments, P-X is selected from:2344429.601_P18646-02X

[0195] In particular embodiments, X is selected from18F,124I,125I,131I, and211At.

[0196] In certain embodiments, C comprises a chelating agent selected from DOTAGA (1,4,7,10-tetraazacyclododececane,l- (glutaric acid)-4,7,10-triacetic acid). DOTA (1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DOTASA (1,4,7,10-tetraazacyclododecane-l-(2-succinic acid)-4,7,10-triacetic acid), CB-DO2A (10-bis(carboxymethyl)-l,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA (7-[2-(Bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-l,4,7,10-tetraaza-cyclododec-l-yl-acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)] [5 -(4-nitrophenyl- 1 - [4,7, 10-tris(carboxymethyl)- 1,4,7, 10-tetraazacyclododecan-l-yl]pentan-2-yl)amino]acetic acid)), TCMC (2-(4-isothiocyanotobenzyl)- 1.4.7.10-tetraaza-l,4,7,10-tetra-(2-carbamonyl methyl)-cyclododecane), oxo-DO3A (1-oxa- 4.7.10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid), p-NIfc-Bn-Oxo-DO3A (l-Oxa-4,7,10-tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((l,8-A, A'-bis-(carboxymethyl)-l,4,8,ll-tetraazacyclotetradecane), MM-TE2A, DM-TE2A, CB-TE2A (4,1 l-bis(carboxymethyl)-l,4,8.1 l-tetraazabicyclo[6.6.2]hexadecane). CB-TE1A1P (4,8,1 l-tetraazacyclotetradecane-l-(methanephosphonic acid)-8-(methanecarboxylic acid), CB-TE2P (l,4,8,ll-tetraazacyclotetradecane-l,8-bis(methanephosphonic acid), TETA (1,4,8,11-2444429.601_P18646-02tetraazacyclotetradecane- 1,4, 8, 11 -tetraacetic acid), NOTA (l,4,7-triazacyclononane-N, N', N"-triacetic acid), NODA (l,4,7-triazacyclononane-l,4-diacetate ); NOD AG A (1,4,7-triazacyclononane.l -glutaric acid-4, 7-acetic acid), (NOTAGA) l,4,7-triazonane-l,4-diyl)diacetic acid DFO (Desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethl-[l,4,7]triazonan-l-yl}-acetic acid), TACN-TM (N, N', N", tris(2-mercaptoethyl)-l,4.7-triazacyclononane), Diamsar (l,8-Diamino-3.6,10.13,16,19-hexaazabicyclo(6.6.6)eicosane, 3.6.10.13.16.19-Hexaazabicyclo[6.6.6]eicosane-l,8-diamine), Sarar (l-A-(4-aminobenzyl)-3, 6.10.13.16.19-hexaazabicyclo[6.6.6] eicosane- 1,8-diamine). AmBaSar (4-((8-amino- 3.6.10.13.16.19-hexaazabicyclo [6.6.6] icosane-l-ylamino) methyl) benzoic acid), macropa, and BaBaSar.

[0197] In certain embodiments, C comprises a chelating agent selected from:^7"'N^CO2H. _, CO2H — N — \ ( CO2H ( CO2H

[0198] C°2HCO2HCO2H2544429.601_P18646-020CO2H

[0203]

[0204] 2644429.601_P18646-02HO O HO O2744429.601_P18646-02

[0209]

[0212] COOH;OH

[00213] 2844429.601_P18646-02

[0217] In certain embodiments, the naturally occurring non-radioactive isotope of a metal is selected from Cu, Pb, Ac, Lu, Ga, Tb, Y, In, Re, Sm, Zr, Bi, Sc, Ho, Ra, Th, and Al. In particular embodiments, the naturally occurring non-radioactive isotope of a metal is selected from27Al,45Sc,63Cu,65Cu,69Ga.71Ga.89Y,90Zr,91Zr,92Zr.94Zr.96Zr,113In.115In.144Sm.147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th.2944429.601_P18646-02

[0218] One of ordinary skilled in the art would appreciate that commercially-available chelating agents can include activating agents, for example, agents that can react with a primary amine. Such agents include, but are not limited to, N-hydroxy succinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), anhydride, maleimide, N-benzyl, 4-isothiocyanatobenzyl (p-NCS-Bz), NH2-MPAA, propargyl, TA. N-(2-aminoethyl)ethanamide, NH2-PEG4, and hydrophilic dPEG spacer bound to a tetrafluorophenyl (TFP) ester. These agents can be bound to or form part of the linker which is bound to the chelating agent.

[0219] Compounds can assembled by reactions between different components, to form linkages such as ureas (-NRC(O)NR-), thioureas (-NRC(S)NR-), amides (-C(O)NR- or -NRC(O)-), or esters (-C(O)O- or -OC(O)-). Urea linkages can be readily prepared by reaction between an amine and an isocyanate, or between an amine and an activated carbonamide (-NRC(O)-). Thioureas can be readily prepared from reaction of an amine with an isothiocyanate. Amides (-C(O)NR- or - NRC(O)-) can be readily prepared by reactions between amines and activated carboxylic acids or esters, such as an acyl halide or N-hydroxy succinimide ester. Carboxylic acids may also be activated in situ, for example, with a coupling reagent, such as a carbodiimide, or carbonyldiimidazole (CDI). Esters may be formed by reaction between alcohols and 20 activated carboxylic acids. Triazoles are readily prepared by reaction between an azide and an alkyne, optionally in the presence of a copper (Cu) catalyst.

[0220] Suitable linkers are disclosed in U. S. Patent Application Publication No. US2011 / 0064657 Al, for " Labeled Inhibitors of Prostate Specific Membrane Antigen (PSMA), Biological Evaluation, and Use as Imaging Agents," published March 17, 2011, to Pomper et al., and U. S. Patent Application Publication No. US2012 / 0009121 Al, for " PSMA-Targeting Compounds and Uses Thereof," published January 12, 2012, to Pomper et al, each of which is incorporated by reference in its entirety.

[0221] In certain embodiments, Laand Lb are each individually selected from (a), (b), (c). or (d):

[0222] (a)

[0223] wherein:

[0224] pi, p2, ps and p4 may be in any order;3044429.601_P18646-02

[0225] t1 and t2 are each an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0226] pi, ps, and p4 are each independently 0 or 1;

[0227] p2 is an integer selected from 0, 1, 2, and 3. and when p2 is 2 or 3, each Ri is the same or different;

[0228] m1, m2, m3, and m4 are each an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8;

[0229] Wi is selected from a bond, -S-, -C(=O)-, -C(=O)-NR-, and -NR-C(=O)-;

[0230] W2 is selected from a bond, -S-, -CH2-C(=O)-NR-, -C(=O)-, -NRC(=O)-, -NR'C(=O)NR-, -NRC(=S)NR'2-, -NRC(=O)O-, -OC(=O)NR-, -OC(=O)-, -C(=O)NR-, -NR-C(=O)—, -C(=O)O-, -(O-CH2-CH2)q- and -(CH2-CH2-O)q, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0231] each R or R' is independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl,

[0232] Y can be present or absent and when present is selected from a nitrogen-containing heteroalkylene chain, a cyclic or bicylic heteroalkyl radical, and a triazole radical having the N N INstructureor

[0233] each Ri is independently H, Ci-Ce alkyl, C3-C12 aryl, -(CH2)q-C3-C12 aryl, -C4-C16 alkylaryl, or -(CH2)q-C4-C16 alkylaryl;

[0234] R2 and R3 are each independently H, -(CH2)q-C3-C12 aryl, and -CO2R5, wherein R5 is selected from H, Ci-Ce alkyl, C3-C12 aryl, and C4-C16 alkylaryl, wherein R2 and R3 can be the same or different, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0235] V is selected from -C(O)-, -C(S)-. -NRC(O)-, -NRC(S)-, and -OC(O)-;R1NW121

[0236] (b)

[0237] pi, p2, ps and p4 may be in any order;3144429.601_P18646-02

[0238] t2 is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0239] pi and ps are each independently 0 or 1;

[0240] p2 is an integer selected from 0, 1, 2, and 3. and when p2 is 2 or 3, each Ri is the same or different;

[0241] mi and m2 are each an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8;

[0242] W2 is selected from a bond, -S-, -CH2-C(=O)-NR-, -C(=O)-, -NRC(=O)-, -NR'C(=O)NR-, -NRC(=S)NR'2-, -NRC(=O)O- -OC(=O)NR-, -OC(=O)-, -C(=O)NR-, -NR-C(=O)—, -C(=O)O-, -(O-CH2-CH2)q- and -(CH2-CH2-O)q, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0243] each R or R' is independently H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocyclo alkyl;

[0244] each R1 is independently H, C1-C6 alkyl, C3-C12 aryl, -(CH2)q-C3-C12 aryl, -C4-C16 alkylaryl, or -(CH2)q-C4-C16 alkylaryl, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0245] R2 and R3 are each independently H, -(CH2)q-C3-C12 aryl, and -CO2R5, wherein R5 is selected from H, C1-C6 alkyl, C3-C12 aryl, and C4-C16 alkylaryl, wherein R2 and R3 can be the same or different, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; and

[0246] V is selected from -C(O)-, -C(S)-, -NRC(O)-, -NRC(S)-, and -OC(O)-;

[0247] (c) -Li-, -L2-L3-, or -L1-L2-L3-, wherein:

[0248] Li is -NR-(CH2)q-[O-CH2-CH2-O]q-(CH2)q-C(=O)-;

[0249] L2is -NR-(CH2)q-C(COOR5)-NR-; and

[0250] L3is -(O=)C-(CH2)q-C(=O)-;

[0251] wherein each q is independently an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0252] R is selected from H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl; and

[0253] R5 is selected from H, C1-C6 alkyl, C3-C12 aryl, and C4-C16 alkylaryl; and

[0254] (d) -(CR6H)q-(CH2)q-C(=O)-NR-(CH2)q-O- or -NR-(CH2)q-O-; wherein:

[0255] R6is H or-COOR5;3244429.601_P18646-02

[0256] each q is independently an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0257] R is selected from H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl; and

[0258] R5 is selected from H, C1-C6 alkyl, C3-C12 aryl, and C4-C16 alkylaryl.

[0259] In certain embodiments, Y is selected from:(R-16)Z4bX1 Z2

[0260] ; wherein Xi and X2 are each independently -CH- or N; each Ri6 is independently H or -C(=O)-ORi7, wherein R17 is C1-C4 alkyl;

[0261] -N(Ri8)-(CH2)z5-N(Ri9)-; wherein Ris and R19 are each independently H or C1-C4 alkyl and Z5 is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[00263]

[0264] In some embodiments, one or more of Laand Lb include one or more units selected from:3344429.601_P18646-02RCOOCH3

[0265] 3444429.601_P18646-02

[0268] wherein u is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0269] each R is independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl; and

[0270] R5 is selected from H, C1-C6 alkyl, C3-C12 aryl, and C4-C16 alkylaryl.

[0271] In particular embodiments, the FAP-alpha binding moiety comprises:

[0273] In particular embodiments, P-X comprises a halogenated fluoronicotinamide:

[0274] N

[0275] In particular embodiments, P-X is 6-[18F]fluoronicotinamide:O— II l| H *

[00276] 18F N

[0277] In particular embodiments, the chelator comprises 1,4,7, 10-tetraazacyclododecane- 1.4,7- tris-acetic acid- 10 monoamide (DOTA-monoamide):3544429.601_P18646-02HOOCHOOC N NN—?HrA

[00278] HOOC-7°

[0279] In particular embodiments, the linker, Lb, comprises a C1-C8 alkylene chain.

[0280] In particular embodiments, the linker, La, comprises:

[0281] o

[0282] In particular embodiments, the La-(C)-Lb-P-X comprises:XcL|? HA_N^-^Oy

[00283] o

[0284] In certain embodiments, the compound of formula (I) is selected from:3644429.601_P18646-02

[0292] 3744429.601_P18646-02

[0294] o3844429.601_P18646-02

[0300]

[0301]

[0302]

[0303]

[0304] and

[0305] In certain embodiments, the compound of formula (I) is selected from3944429.601_P18646-0264044429.601_P18646-02

[0312]

[0313]

[0314]

[0315]

[0316]

[00317] 4144429.601_P18646-02

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] 4244429.601_P18646-02

[0326] wherein M is a naturally occurring non-radioactive isotope of a metal.

[0327] In certain embodiments, the compound of formula (I) is selected from:4344429.601_P18646-02

[0333]

[0336] 4444429.601_P18646-0244429.601_P18646-02

[0348] wherein M is a naturally occurring non-radioactive isotope of a metal.

[0349] In particular embodiments, the compound of formula (I) comprises:4644429.601_P18646-02

[00350]

[0351] wherein M is selected from113In / 115In,45Sc, and69Ga / 71Ga.

[0352] In more particular embodiments, the compound of formula (I) comprises:

[0354] wherein M is69Ga / 71Ga.

[0355] B. Methods of Synthesizing Compounds of Formula [1SF]-M-1

[0356] In other embodiments, the presently disclosed subject matter provides a method for synthesizing:

[0358] the method comprising:

[0359] (a) providing a trimethylammonium precursor compound (3):

[0361] (b) contacting the trialkylammonium precursor compound (3) with18F / 4,7,13,16,21,24- hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane in the presence of a base in a polar, aprotic solvent at a first tempearture to form [18F] 1:4744429.601_P18646-02

[0363] (c) contacting ([18F] 1) with M3+in a buffer at a second temperature to form

[0365] wherein M is a naturally occurring non-radioactive isotope of a metal.

[0366] In certain embodiments, M is selected from Cu, Pb, Ac, Lu, Ga, Tb, Y, In, Re, Sm, Zr, Bi, Sc, Ho, Ra, Th, and Al. In particular embodiments, M is selected from27Al,45Sc,63Cu,65Cu,69Ga,71Ga,89Y,90Zr.91Zr.92Zr,94Zr,96Zr,113In,115In.144Sm.147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th. In more particular embodiments, M is selected from113 / 115In,45Sc, and69Ga / 71Ga.

[0367] In certain embodiments, M3+is a salt selected from113In / 115InCl3,45ScCl3,113In / 115In (NO3)3,45SC(NO3)3, and69Ga / 71Ga(NO3)3.

[0368] In certain embodiments, M is a naturally occurring non-radioactive isotope or mixture of isotopes of a metal. In such embodiments, the naturally occurring metal isotopes are designated asnatM.

[0369] More particularly, in such embodiments. M is selected fromnatCu,natPb,natAc,natLu,natGa,natTb,natY,natIn,natRe,natSm,natZr,natBi,natSc,natHo,natRa,natTh, andnatAl. In particular embodiments, M is selected from27Al,45Sc,63Cu,65Cu,69Ga,71Ga,89Y,90Zr,91Zr,92Zr,94Zr,96Zr,113In,113In,144Sm,147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb.175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th. In more particular embodiments, M is selected fromnatIn,natSc, andnatGa. In certain embodiments, M3+is a salt selected fromnatInCl3,natScCl3,natGaC13,natIn(NO3)3.natSc(NO3)3. andnatGa(NO3)3.

[0370] One of ordinary skill in the art would appreciate, for example, the most abundant natural isotope of indium,115In, is only a very slightly radioactive isotope decaying by beta decay with a4844429.601_P18646-02half-life of 4.41×1014years. For the purposes of this disclosure, this natural radioactivity is negligible.

[0371] In certain embodiments, the base is potassium oxalate. In certain embodiments, the polar, aprotic solvent comprises dimethylsulfoxide (DMSO). In certain embodiments, the first temperature is about 90 °C. In certain embodiments, the buffer is an acetate buffer. In certain embodiments, step (c) is conducted at a pH of about 5. In certain embodiments, the second temperature is about 70 °C.

[0372] In certain embodiments, [18F]-M-1 is purified by preparative high performance liquid chromatography. In certain embodiments, the method further comprises purifying [18F]-M-1 by solid-phase extraction (SPE).

[0373] In certain embodiments, [18F]-M-1 has a radiochemical yield of about 25%. In certain embodiments, [18F]-M-1 has a radiochemical purity greater than about 95%. In certain embodiments, [18F]-M-1 has a molar (specific) radioactivity between about 2000 to about 5000 Ci / mmol.

[0374] Pharmaceutical Compositions

[0375] In some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising the compound of formula (I). In certain embodiments, the formulation comprises one or more of pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. In such embodiments, the present disclosure provides a pharmaceutical composition including a compound of formula (I) alone or in combination with one or more additional therapeutic agents in admixture with a pharmaceutically acceptable excipient.

[0376] One of skill in the art will recognize that the pharmaceutical compositions include the pharmaceutically acceptable salts of the compounds described above. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and include salts of active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base4944429.601_P18646-02addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.

[0377] When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example. Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0378] Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20thed.) Lippincott, Williams & Wilkins (2000). In therapeutic and / or diagnostic applications, the compounds of the disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington: The Science and Practice of Pharmacy (20thed.) Lippincott, Williams & Wilkins (2000).5044429.601_P18646-02

[0379] Depending on the specific conditions being treated, such agents may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20thed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articullar, intra -sternal, intra-synovial, intra- hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

[0380] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0381] Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0382] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.

[0383] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those5144429.601_P18646-02skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A nonlimiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the compound(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound(s), and the preference and experience of the attending physician.

[0384] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0385] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethyl-cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0386] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic. talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0387] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as5244429.601_P18646-02lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

[0388] Methods for Imaging

[0389] In other embodiments, the presently disclosed subject matter provides a method for imaging a disease or disorder associated with fibroblast-activation protein-a (FAP-a), the method comprising administering a compound of formula (I), or a pharmaceutical composition thereof, and obtaining an image.

[0390] In other embodiments, the presently disclosed subject matter provides a method for inhibiting fibroblast-activation protein-a (FAP-a), the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutical composition thereof.

[0391] Accordingly, in some embodiments, the presently disclosed subject matter provides a method for imaging one or more cells, organs, or tissues, the method comprising exposing cells or administering to a subject an effective amount of a compound of formula (I) with a radioisotopic label suitable for imaging. In some embodiments, the one or more organs or tissues include prostate tissue, kidney tissue, brain tissue, vascular tissue, or tumor tissue.

[0392] The imaging methods of the invention are suitable for imaging any physiological process or feature in which FAP-a is involved, for example, identifying areas of tissues or targets which exhibit or express high concentrations of FAP-a.

[0393] Physiological processes in which FAP-a is involved include, but are not limited to: (a) proliferation diseases (including but not limited to cancer); (b) tissue remodeling and / or chronic inflammation (including but not limited to fibrotic disease, wound healing, keloid formation, osteoarthritis, rheumatoid arthritis, and related disorders involving cartilage degradation); and (c) endocrinological disorders (including but not limited to disorders of glucose metabolism).

[0394] In certain embodiments, the radiolabeled compound is stable in vivo.

[0395] In certain embodiments, the radiolabeled compound is detected by positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0396] In certain embodiments, the presently disclosed compounds are excreted from tissues of the body quickly to prevent prolonged exposure to the radiation of the radiolabeled compound5344429.601_P18646-02administered to the subject. Typically, the presently disclosed compounds are eliminated from the body in less than about 24 hours. More typically, the presently disclosed compounds are eliminated from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours. 2 hours, 90 minutes, or 60 minutes. Exemplary compounds are eliminated in between about 60 minutes and about 120 minutes. In certain embodiments, the presently disclosed compounds are stable in vivo such that substantially all, e.g., more than about 50%. 60%. 70%, 80%, or 90% of the injected compound is not metabolized by the body prior to excretion.

[0397] Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids and cell samples of the above subjects will be suitable for use, such as mammalian, particularly primate such as human, blood, urine or tissue samples, or blood urine or tissue samples of the animals mentioned for veterinary applications.

[0398] Other embodiments provide kits comprising a compound of formula (I). In certain embodiments, the kit provides packaged pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of formula (I). In certain embodiments the packaged pharmaceutical composition will comprise the reaction precursors necessary to generate the compound of formula (I) upon combination with a radiolabeled precursor. Other packaged pharmaceutical compositions further comprise indicia comprising at least one of: instructions for preparing compounds of formula (I) from supplied precursors, instructions for using the composition to image cells or tissues expressing FAP-α.

[0399] In certain embodiments, a kit containing from about 1 to about 30 mCi of the radionuclide-labeled imaging agent described above, in combination with a pharmaceutically acceptable carrier, is provided. The imaging agent and carrier may be provided in solution or in lyophilized form. When the imaging agent and carrier of the kit are in lyophilized form, the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. The kit may provide a compound of formula (I) in solution or in lyophilized form, and these components of the kit may optionally contain stabilizers such as NaCl, silicate, phosphate buffers, ascorbic acid, gentisic acid, and the like. Additional stabilization of kit components may be provided in this embodiment, for example, by providing the reducing agent in an oxidation-resistant form. Determination and optimization of such stabilizers and stabilization methods are well within the level of skill in the art.5444429.601_P18646-02

[0400] In certain embodiments, a kit provides a non-radiolabeled precursor to be combined with a radiolabeled reagent on-site.

[0401] Imaging agents may be used in accordance with the presently disclosed methods by one of skill in the art. Images can be generated by virtue of differences in the spatial distribution of the imaging agents which accumulate at a site when contacted with FAP-a. The spatial distribution may be measured using any means suitable for the particular label, for example, a gamma camera, a PET apparatus, a SPECT apparatus, and the like. The extent of accumulation of the imaging agent may be quantified using known methods for quantifying radioactive emissions or fluorescence. A particularly useful imaging approach employs more than one imaging agent to perform simultaneous studies.

[0402] In general, a detectably effective amount of the imaging agent of the invention is administered to a subject. A “detectably effective amount” of the imaging agent is defined as an amount sufficient to yield an acceptable image using equipment which is available for clinical use. A detectably effective amount of the imaging agent may be administered in more than one injection. The detectably effective amount of the imaging agent of the invention can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, and the dosimetry. Detectably effective amounts of the imaging agent also can vary according to instrument and film-related factors. Optimization of such factors is well within the level of skill in the art. The amount of imaging agent used for diagnostic purposes and the duration of the imaging study will depend upon the radionuclide used to label the agent, the body mass of the patient, the nature and severity of the condition being treated, the nature of therapeutic treatments which the patient has undergone, and on the idiosyncratic responses of the patient. Ultimately, the attending physician will decide the amount of imaging agent to administer to each individual patient and the duration of the imaging study.

[0403] Methods for Treating a FAP-a and / or Related Disease or Disorder using the Compounds of Formula (I), or Pharmaceutical Compositions Thereof

[0404] In some embodiments, the presently disclosed subject matter provides a method for inhibiting fibroblast- activation protein-a (FAP-a), the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutical composition thereof.5544429.601_P18646-02

[0405] In other embodiments, the presently disclosed subject matter provides a method for treating a fibroblast-activation protein-a (FAP-a)-related disease or disorder, the method comprising administering to a subject in need of treatment thereof an effective amount of a compound of formula (I), or a pharmaceutical composition thereof, wherein the compound of formula (I) comprises a radiolabeled functional group suitable for radiotherapy.

[0406] Many radionuclides, primarily β- and alpha emitters, have been investigated for targeted radioimmunotherapy and include both radiohalogens and radiometals. Representative therapeutic radionuclides include, but are not limited to: β-particle emitters90Y,1311,177Lu,153Sm,186Re,188Re,67Cu,212Pb; a-particle emitters225Ac,213Bi,212Bi,211At,212Pb; and Auger electron emitters125I,123I,67Ga, andmIn.

[0407] Radioisotopes suitable for use with the presently disclosed subject matter also include, but are not limited to,11C,18F,51Cr,68Ga,99mTc,130La,140La,175Yb,153Sm,166Ho,88Y,149Pm,165Dy,169Er,177LU,47SC,142Pr,159Gd,212Bi,72As,72Se,97Ru,109Pd,105Rh,101mRh,119Sb,128Ba,124I,197Hg,151EU.153EU,169EU,201Tl,203Pb.64Cu,198AU.225AC,227Th, and199Ag.

[0408] In some embodiments, the presently disclosed compounds of formula (I) can be used to treat a subject afflicted with one or more FAP-a related diseases or disorders including, but not limited to: (a) proliferation (including but not limited to cancer); (b) tissue remodeling and / or chronic inflammation (including but not limited to fibrotic disease, wound healing, keloid formation, osteoarthritis, rheumatoid arthritis and related disorders involving cartilage degradation); and (c) endocrinological disorders (including but not limited to disorders of glucose metabolism).

[0409] Accordingly, in some embodiments, the one or more FAP-a related disease or disorder is selected from the group consisting of a proliferative disease, including, but not limited to, breast cancer, colorectal cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, melanoma, fibrosarcoma, bone and connective tissue sarcomas, renal cell carcinoma, giant cell carcinoma, squamous cell carcinoma, gastric cancer, a glioma, and adenocarcinoma, pancreatic ductal adenocarcinoma; diseases characterized by tissue remodeling and / or chronic inflammation; disorders involving endocrinological dysfunction; and blood clotting disorders.

[0410] In general, the “effective amount” of an active agent or drug delivery devicerefers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending5644429.601_P18646-02on such factors as the desired biological endpoint, the agent to be delivered, the makeup of the pharmaceutical composition, the target tissue, and the like.

[0411] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0412] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0413] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration,5744429.601_P18646-02progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0414] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a compound disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0415] Further, the compounds disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0416] The timing of administration of a compound disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a compound described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e„ simultaneously) or at different5844429.601_P18646-02times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0417] When administered sequentially, the agents can be administered within 1, 5, 10, 30. 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0418] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be. additive or synergistic. The agents may be administered multiple times.

[0419] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0420] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0421] Qa / QA+ Qb / QB= Synergy Index (SI)

[0422] wherein:

[0423] Q is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0424] Qais the concentration of component A, in a mixture, which produced an end point;

[0425] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and5944429.601_P18646-02

[0426] Qb is the concentration of component B, in a mixture, which produced an end point.

[0427] Generally, when the sum of Qa / QAand Qb / QBis greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one. synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0428] Depending on the specific conditions being treated, the “agent(s)” may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra- sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

[0429] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0430] Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be6044429.601_P18646-02formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0431] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol: absorption promoters; and fluorocarbons.

[0432] In particular embodiments, the compound disclosed herein is administered intranasally in a form selected from a nasal spray, a nasal drop, a powder, a granule, a cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures.

[0433] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the compound(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound(s), and the preference and experience of the attending physician.

[0434] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0435] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.6144429.601_P18646-02Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0436] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0437] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

[0438] Further, one of ordinary skill in the art will recognize that the presently disclosed compounds, and pharmaceutical compositions thereof, include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and include salts of active compounds that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. The parent form of the compound can differ from the various salt forms in certain physical properties, such as solubility, and the like.

[0439] When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like.6244429.601_P18646-02

[0440] When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See. for example, Berge et al. “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Compounds containing both basic and acidic functionalities allow such compounds to be converted into either base or acid addition salts.

[0441] Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, monohydrogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, galactonate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate, monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate, succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (-i-)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p-toluenesulfonate and trifluoroacetate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0442] Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the " Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0443] The term “hydrocarbon” as used herein, refers to any chemical group comprising hydrogen and carbon. A hydrocarbon group may be substituted or unsubstituted. As would be known to one of ordinary skill in the art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.6344429.601_P18646-02

[0444] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 alipatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0445] The term “alkane” refers to acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, and therefore consisting entirely of hydrogen atoms and saturated carbon atoms.

[0446] The term “alkyl” refers to a univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom and having the chemical formula of -CnH2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R ≠ H), and R3C (R ≠ H) are primary, secondary and tertiary alkyl groups, respectively.

[0447] An alkyl can be a straightchain (i.e., unbranched) or branched acyclic hydrocarbon having the number of carbon atoms designated (i.e., C1-10 means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons). In particular embodiments, the term “alkyl” refers to C1-20 inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. In other embodiments, the alkyl can be a C1-C4 alkyl, including 1, 2, 3, and 4 carbons. In yet other embodiments, the alkyl can be a C1-C6alkyl, including 1, 2, 3, 4, 5, and 6 carbons. In even yet other embodiments, the alkyl can be a C1-C8alkyl, including 1, 2, 3, 4, 5, 6, 7, and 8 carbons.6444429.601_P18646-02

[0448] “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers to straight-chain alkyls. In other embodiments, “alkyl” refers to branched alkyls. In certain other embodiments, “alkyl” refers to straight-chain and / or branched alkyls. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.

[0449] Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, w-butyl, isobutyl, sec-butyl, tert-butyl, w-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[0450] Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more substituents, which can be the same or different. Such substituent groups include, but are not limited to, alkyl, substituted alkyl, cycloalkyl, halogen, acyl, carboxyl, oxo, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto.

[0451] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain having from 1 to 20 carbon atoms or heteroatoms consisting of at least one carbon atom and at least one heteroatom selected from O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0452] The term “cycloalkane” refers to saturated monocyclic hydrocarbons (with or without side chains), e.g., cyclobutane. Unsaturated monocyclic hydrocarbons having one endocyclic double or one triple bond are called cycloalkenes and cycloalkynes, respectively. Those having more than one such multiple bond are cycloalkadienes, cycloalkatrienes, and the like. The inclusive terms for any cyclic hydrocarbons having any number of such multiple bonds are cyclic olefins or cyclic acetylenes.6544429.601_P18646-02

[0453] The term “cycloalkyl” refer to a univalent group derived from a cycloalkane by removal of a hydrogen atom from a ring carbon atom. Cycloalkyls can be a mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g.. 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group also can be optionally substituted with a substituent group provided hereinabove for alkyl groups. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like.

[0454] The term “cycloalkylalkyl” as used herein, refers to a cycloalkyl group, which is attached to the parent molecular moiety through an alkylene moiety, also as defined above, e.g., a C1-20 alkylene moiety. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0455] The terms “cycloheteroalkyl” and “heterocycloalkyl” (or more generally “heterocyclic”) are used interchangeably and refer to an unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), in which the nitrogen, sulfur, and phosphorus heteroatoms may be oxidized and the nitrogen heteroatom may be quaternized. The cyclohetero alkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0456] The terms “cycloalkylene” and “heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0457] As used herein the terms “bicycloalkyl” and “bicycloheteroalkyl” refer to two cycloalkyl or cycloheteroalkyl groups that are bound to one another. Non-limiting examples include bicyclohexane and bipiperidine.

[0458] An “unsaturated hydrocarbon” has one or more double bonds or triple bonds. As used herein, the term “alkene” refers to an acyclic branched or unbranched hydrocarbons having one6644429.601_P18646-02carbon-carbon double bond and the general formula CnH2n. Acyclic branched or unbranched hydrocarbons having more than one double bond are alkadienes, alkatrienes, and the like.

[0459] More particularly, the term “alkenyl” as used herein refers to a monovalent group derived from a C2-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen molecule. Alkenyl groups include, but are not limited to. ethenyl (i.e., vinyl), 2-propenyl, butenyl, l-methyl-2-buten-l-yl, pentenyl, 2-isopentenyl, hexenyl, octenyl, allenyl, butadienyl, crotyl (but-2-en-l-yl), 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), and the like, including higher homologs and isomers.

[0460] The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

[0461] The term “alkyne” as used herein refers to an acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula CnH2n-2. RC=CR. Acyclic branched or unbranched hydrocarbons having more than one triple bond are known as alkadiynes, alkatriynes, and the like.

[0462] The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C2-20 hydrocarbon of a designed number of carbon atoms containing at least one carboncarbon triple bond. Examples of alkynyl groups include, but are not limited to. ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and heptynyl groups, and the like.

[0463] As used herein, the term “alkylene” refers to an alkanediyl group having the free valencies on adjacent carbon atoms, e.g. -CH(CH3)CH2- propylene (systematically called propane-1, 2-diyl). More particularly, the term “alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1. 2, 3, 4, 5, 6, 7. 8, 9, 10, 11. 12, 13. 14, 15. 16. 17. 18. 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (–(CH2)3–); cyclohexylene (–C6H10–); –CH=CH–CH=CH–;6744429.601_P18646-02–CH=CH–CH2–; –CH2CH2CH2CH2–, –CH2CH=CHCH2–, –CH2C≡CCH2–, CH2CH2CH(CH2CH2CH3)CH2–, –(CH2)q–N(R)–(CH2)r–, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16. 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-O-CH2-O-); and ethylenedioxyl (–O–(CH2)2–O–). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0464] The term “hetero alkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms also can occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and -R’OC(O)-.

[0465] The term “arene” refers to a monocyclic and polycyclic aromatic hydrocarbon.

[0466] The term “aryl” refers to a group derived from arenes by removal of a hydrogen atom from a ring carbon atom. Groups similarly derived from heteroarenes are sometimes subsumed in this definition. An aryl group can include, for example, a single ring or multiple rings (such as from 2 to 3 rings), which are fused together or linked covalently.

[0467] The term “heteroaryl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m(l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group.

[0468] The term “heteroaryl” refers to the class of heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom from any ring atom. A “heteroaryl” group can include from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. A heteroaryl group can be attached to the remainder of the molecule6844429.601_P18646-02through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl. 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl. 2-phenyl-4-oxazolyl. 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl. 1 -isoquinolyl. 5- isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms “arylene” and “heteroarylene” refer to the divalent forms of aryl and heteroaryl, respectively.

[0469] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms “arylalkyl” and “heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like). However, the term “haloaryl,” as used herein is meant to cover only aryls substituted with one or more halogens.

[0470] Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g. “3 to 7 membered”), the term “member” refers to a carbon or heteroatom.

[0471] Each of above terms defined hereinabove (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and “heterocycloalkyl”, “alkenyl”, “alkynyl,” “aryl,” “heteroaryl,” as well as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below.

[0472] As used herein, the term “acyl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m(l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group. For example, in some embodiments, the term acyl includes an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetyl group.6944429.601_P18646-02Specific examples of acyl groups include acetyl and benzoyl. Acyl groups also are intended to include amides, -RC(=O)NR’, esters, -RC(=O)OR’, ketones, -RC(=O)R’, and aldehydes, -RC(=O)H.

[0473] The terms “alkoxy!” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O-) or unsaturated (i.e., alkenyl-O- and alkynyl-O-) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.

[0474] The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

[0475] “Aryloxyl” refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

[0476] “Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0477] “Aralkyloxy 1” refers to an aralkyl-O- group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl, i.e., C6H5-CH2-O-. An aralkyloxyl group can optionally be substituted.

[0478] “Alkoxycarbonyl” refers to an alkyl-O-C(=O)- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and tert-butyloxycarbonyl.

[0479] “Aryloxycarbonyl” refers to an aryl-O-C(=O)- group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0480] “Aralkoxycarbonyl” refers to an aralkyl-O-C(=O)- group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0481] The term “acyloxyl” refers to an oxygen-centered radicals consisting of an acyl radical bonded to an oxygen atom, e.g., an acyl-O- group wherein acyl is as previously described.

[0482] The term “amine” refers to a compound formally derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbyl groups, and having the general structures RNH27044429.601_P18646-02(primary amines), R2NH (secondary amines), R3N (tertiary amines). In some embodiments, the term amino refers to the -NH2 group. More generally, the amino group is -NR'R”, wherein R' and R” are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0483] The terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.

[0484] An “aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure -NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure -NR’R”, wherein R’ and R” are each independently selected from alkyl groups. The term trialkylamino refers to a group having the structure -NR’ R”R”’, wherein R’, R”, and R’” are each independently selected from alkyl groups. Additionally, R’, R”, and / or R’” taken together may optionally be -(CH2)k- where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino, trimethylamino, and propylamino.

[0485] The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S-) or unsaturated (i.e., alkenyl-S- and alkynyl-S-) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0486] “Acylamino” refers to an acyl-NH- group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH- group wherein aroyl is as previously described.

[0487] The term “carbonyl” refers to a compound containing the carbonyl group, -C(=O)-. The term is commonly used in the restricted sense of aldehydes (R-C(=O)H) and ketones, although it actually includes carboxylic acids and derivatives.

[0488] The term “carboxylic acid” refers to an oxoacids having the structure RC(=O)OH. The term is used as a suffix in systematic name formation to denote the -C(=O)OH group including its7144429.601_P18646-02carbon atom. Tn some embodiments, the term “carboxyl” refers to the -COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.

[0489] “Carbamoyl” refers to an amide group of the formula -C(=O)NH2.

[0490] “Alkylcarbamoyl” refers to a R’RN-C(=O)- group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and / or substituted alkyl as previously described.

[0491] “Dialkylcarbamoyl” refers to a R’RN-C(=O)- group wherein each of R and R’ is independently alkyl and / or substituted alkyl as previously described.

[0492] The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula -O-C(=O)-OR.

[0493] The term “cyano” refers to the -C=N group.

[0494] The terms “halo.” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(Ci-4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0495] The term “hydroxyl” refers to the -OH group.

[0496] The term “hydroxyalkyl” refers to an alkyl group substituted with an -OH group.

[0497] The term “mercapto” refers to the -SH group.

[0498] The term “oxo compound” refers to a compounds containing an oxygen atom, =0, doubly bonded to carbon or another element. The term thus embraces aldehydes, carboxylic acids, ketones, sulfonic acids, amides and esters. Oxo used as an adjective (and thus separated by a space) modifying another class of compound, as in oxo carboxylic acids, indicates the presence of an oxo substituent at any position. To indicate a double-bonded oxygen that is part of a ketonic structure, the term keto is sometimes used as a prefix, but such use has been abandoned by TUPAC for naming specific compounds. A traditional use of keto is for indicating oxidation of CHOH to C=O in a parent compound that contains OH groups, such as carbohydrates, e.g. 3-ketoglucose. In some embodiments, the term “oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.

[0499] The term “nitro” refers to the -NO2 group.

[0500] The term “thio” refers to replacement of an oxygen by a sulfur, e.g., PhC(=S)NH2, thiobenzamide.7244429.601_P18646-02

[0501] The term “thiol” refers to a compounds having the structure RSH (R H), e.g., MeCFESH ethanethiol. A thiol also is known by the term “mercaptan.”

[0502] The term “thiohydroxyl” or “thiol,” as used herein, refers to a group of the formula -SH.

[0503] The term “sulfate” refers to the -SO4 group.

[0504] The term “sulfide” refers to a compound having the structure RSR (R ≠ H) and also are referred to as “thioethers.”

[0505] The term “sulfone” refers to a compound having the structure, RS(=O)₂R (R ≠ H), e.g., C2H5S(=O)2CH3 ethyl methyl sulfone.

[0506] The term “sulfoxide” refers to a compound having the structure R₂S=O (R ≠ H), e.g., Ph2S=O diphenyl sulfoxide.

[0507] The term “ureido” refers to a urea group of the formula -NH — CO — NH2.

[0508] One of ordinary skill in the art would recognize that a structure represented generally by, for example, the formula:— rr< R)n

[0509]

[0510] as used herein refers to a ring structure, for example, but not limited to a 3-carbon, a 4- carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and / or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:7344429.601_P18646-02

[00511] R2

[0512] and the like.

[0513] A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.

[0514] The symbol ( ) denotes the point of attachment of a moiety to the remainder of the molecule.

[0515] When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond.

[0516] Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

[0517] Certain compounds of the present disclosure may possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as D- or L- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and / or isolate. The present disclosure is meant to include compounds in racemic, scalemic, and optically pure forms. Optically active (R)- and (S)-, or D- and L-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[0518] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center.7444429.601_P18646-02Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0519] As used herein, the term “congener” refers to one of two or more substances related to each other by origin, structure, or function.

[0520] The term “enantiomer” refers to one of a pair of molecular entities which are mirror images of each other and non-superposable.

[0521] The term “stereoisomer” refers to an isomer that possess identical constitution, but which differ in the arrangement of their atoms in space.

[0522] The term “racemate” refers to an equimolar mixture of a pair of enantiomers. It does not exhibit optical activity. The chemical name or formula of a racemate is distinguished from those of the enantiomers by the prefix (±)- or rac- (or racem-) or by the symbols RS and SR.

[0523] The term “diastereoisomerism” refers to stereoisomerism other than enantiomerism. Diastereoisomers (or diastereomers) are stereoisomers not related as mirror images. Diastereoisomers are characterized by differences in physical properties, and by some differences in chemical behavior towards achiral as well as chiral reagents.

[0524] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0525] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures with the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0526] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon- 14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0527] The term “about.” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies7544429.601_P18646-02that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / - 15%, + / -10%, + / -5%, + / -4%, + / -3%, + / -2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3. 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0528] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0529] The terms “comprise(s) “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0530] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 17644429.601_P18646-02

[0531] 18F-FPyQCP, a PFT Imaging Agent for Detecting Fibroblast Activation Protein

[0532] Overview

[0533] This Example provides18F-FPyQCP, a high-affinity, FAP-targeted PET agent, which demonstrates promising in vivo performance in preclinical studies, supporting clinical translation. Fibroblast activation protein-a (FAP) is a pan-cancer imaging biomarker due to its overexpression in cancer-associated fibroblasts present within 90% of epithelial tumors. Current18F-labeled. FAP-targeted agents for PET have primarily leveraged aluminum fluoride to chelate the radioisotope, with Al18F-FAPI-74 being the most clinically advanced. We aimed to develop a selective18F-labeled, FAP-targeted agent by introducing a 6-fluoronicotinamide precursor to the previously reported (4-quinolinoyl)-glycyl-2-cyanopyrrolidine-based small-molecules, FAPI-04 and QCP02. Without wishing to be bound to any one particular theory, it was thought that an agent employing a18F-6-fluoronicotinamide would produce a stable compound in high yield and molar activity with pharmacokinetics suitable for translation. Compound 1 was designed to carry a metal chelator and the precursor to 6-fluoronicotinamide to introduce the18F radionuclide. Initially, we assessed radiometal analogs In-l and68Ga-l. Subsequently,18F-labeled compounds,18F-1,18F-natIn-l, and18F-natGa-l (18F-FPyQCP), were synthesized in high yields in a one-pot, semi- automated method.18F-FPyQCP demonstrated significantly higher tumor uptake and tumor-to-blood ratios than18F-1 and18F-natIn-l and18F-natSc-l in experimental murine models. Further head-to-head comparisons of18F-FPyQCP and Al18F-FAPI-74 in three human xenograft models with varying levels of FAP expression demonstrated that18F-FPyQCP achieved significantly higher tumor uptake alongside improved biodistribution. Specifically, biodistribution and PET imaging revealed rapid blood and normal tissue clearance for18F-FPyQCP with lower accumulation in the stomach and gastrointestinal tract compared to Al18F-FAPI-74. PET imaging and dosimetry in a healthy non-human-primate confirmed similar absorbed doses for18F-FPyQCP and Al18F-FAPI-74 to most normal tissues. These results support the clinical translation of18F-FPyQCP across various tumors.

[0534] Background

[0535] The explosion of new work in and patient demand for molecular radiotherapy (theranostics) suggests its utility in enhancing quality and potentially prolongation of life. Generally, the first step in implementing a new theranostic agent is to localize and quantify its distribution spatially using the corresponding imaging agent to ensure target engagement while7744429.601_P18646-02avoiding off-target tissues. Few theranostics have been approved by the FDA, however, and none targeting the fibroblast activation protein alpha (FAP-a, FAP), which is a particularly promising target both for localizing and ultimately treating a variety of malignancies. FAP is a dimeric, 170-kDa cell surface serine protease that has exopeptidase and endopeptidase / gelatinase / collagenase activity. Brennen et al., 2012; Abella et al., 2018.

[0536] FAP is abundantly expressed in more than 90% of reactive stromal fibroblasts of human epithelial cancers and has limited expression in normal, healthy tissues or adult tissues or fibroblasts of benign epithelial tumors. Kalluri, 2016; Garin-Chesa et al., 1990. FAP, also called seprase, is an independent adverse prognostic factor for several malignancies, including colorectal, pancreatic, hepatocellular, and ovarian cancer. Henry et al., 2007; Fitzgerald and Weiner, 2020; Mentlein et al., 2011; Hoppner et al., 2023.

[0537] Several studies have shown that FAP-based imaging with positron emission tomography (PET) is a promising noninvasive and quantitative tool for the detection and therapeutic monitoring of cancer. Hoppner et al., 2023; Mona et al., 2022; Kratochwil et al., 2019; Koerber et al., 2020. FAP-targeted imaging agents have emerged as pan-cancer diagnostic tools, comparing favorably with18F-FDG in detecting numerous tumor types. Kratochwil et al., 2019; Chen et al., 2020; Kline et al., 2024. Furthermore, FAP-targeted PET imaging correlates with higher grade lesions, lymph node involvement, and lower overall survival for gastric cancer, glioma, ovarian cancer, and pancreatic ductal adenocarcinoma. Koerber et al., 2020; Pang et al., 2021; Röhrich et al., 2019; Windisch et al., 2020.

[0538] Several FAP-targeting small molecule agents have been translated clinically for PET imaging employing the 4-quinolinoyl-glycyl-2-cyanopyrrolidine pharmacophore. Jansen et al., 2013; Jansen et al., 2014. Among them, those employing68Ga-DOTA have garnered attention due to the potential for easily switching to a therapeutic nuclide. Greifenstein et al., 2023; Loktev et al., 2019; Fendler et al., 2022; Baum et al., 2022; Kelly et al., 2021; Watabe et al., 2020. The development of18F-labeled compounds in high molar activity would be more desirable, however, because18F has favorable physical characteristics, including a high positron decay ratio (97%), relatively short half-life (109.7 min), low positron energy (maximum 0.635 MeV), and a short positron diffusion range (less than 2.4 mm), enabling images of higher resolution than from68Ga. The longer physical half-life of18F compared to68Ga (t1 / 2= 67.7 min) allows for facile transport to satellite imaging facilities that lack onsite cyclotron-based radionuclide production. Moreover,7844429.601_P18646-02cyclotron-based production of18F is suggested to be more cost-effective than generator-based production of68Ga in the current clinical setting. Sahnoun et al., 2020.

[0539] The most extensively studied18F-labeled, FAP-targeted investigational agents include18F-AlF-FAPI-42 and18F-A1F-FAPL74 (FIG. 1). Giesel et al., 2021; Lindner et al., 2021; Wang et al., 2021; Witek et al., 2024. In those compounds, the18F radionuclide is bound to aluminum through an Al-F coordinate bond in a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator. Others have published similar analogs with modified linkers, for example,18F-A1F-P-FAPI and18F-A1F-FAPT. Hu et al., 2022; Huang et al., 2022.

[0540] A limited number of18F-labeled agents also have also been developed in which the18F is covalently linked through a C-F bond to the FAP targeting moiety using various non-metalated prosthetic groups. Toms et al., 2020; Poulie et al., 2023. Furthermore, Yang et al. developed a theranostic agent containing a silicon-based fluoride acceptor (SiFA) prosthetic group with a covalent18F and DOTAGA moiety in a single molecule. Yang et al., 2023. That agent proved capable of imaging FAP-expressing tumor in relevant experimental models, but the uptake in other organs was high, perhaps due to the highly hydrophobic nature of SiFA. Despite tumor-specific uptake, most published compounds do not perform as well as the leading chelator-based agents, such as18F-AlF-FAPI-74.

[0541] To overcome the issues with current FAP-based PET imaging agents and considering the increased importance of FAP-based PET imaging in oncology, we assessed three new compounds that employ a 6-fluoronicotinamide moiety to introduce18F. That prosthetic group has been leveraged to produce18F-labeled compounds in high yield and molar activity in an industrial scale preparation in a single step using readily available intermediates. U.S. Patent No. 10947197B2 for Synthesis of the radiolabeled prostate-specific membrane antigen (PSMA) inhibitor [18F]DCFPyL, to Ravert et al., issued March 16, 2021, which is incorporated herein by reference in its entirety; and Rong et al., 2023.

[0542] Additionally, in vivo metabolic stability of such labeled analogs has been well-documented in patients. Unlike the hydrophobic prosthetic groups fluorobenzamide or SiFA, the 6-fluoronicotinamide moiety is hydrophilic and does not promote high non-specific binding. Notably, an approved prostate-specific membrane antigen (PSMA)-based PET imaging agent,18F-DCFPyL (piflufolastat F18), utilizes the same prosthetic group. Szabo et al., 2015.7944429.601_P18646-02

[0543] In this Example, we selected the high affinity FAP-targeted small molecule aminoquinolines QCP2 and FAPI-04 as agents to create the18F-labeled compounds in this Example (FIG. 2). Loktev et al., 2019; Slania et al., 2021; Boinapally et al., 2022.

[0544] We showed that those18F-labeled agents required that the polarity of the prosthetic group proved favorable, as evidenced by our initial111In- / 68Ga-labeled analogs. Furthermore, the18F- labeled intermediates demonstrated high stability during the metal complexation step using stable / natural isotope (nat)natInC13 andnatGaC13. One compound,18F-FPyQCP, was further evaluated in relevant experimental models with variable FAP expression in mice and was compared head-to-head with18F-AlF-FAPI-74 in tissue biodistribution and PET imaging studies in mice. Finally, we tested18F-FPyQCP in PET imaging and radiation dosimetry studies in a healthy non-human-primate, confirming that the agent performed similarly to18F-AlF-FAPI-74. One aspect in optimization toward18F-FPyQCP was leveraging the chelator to enhance pharmacokinetics by testing natural metals of different diameters and charges, rather than using the chelator for radionuclide binding.

[0545] Results

[0546] Lead optimization - radiosynthesis revealed higher yields and purity for18F-FPyQCP and18F-natIn-1 compared to18F-1

[0547] Compound 1 was designed based on the structure of QCP2 by attaching a small bifunctional linker, L-2,4-aminobutyric acid, to generate compound 2 as shown in Scheme 1.HAm ™.,NHFmoc H<NXCNScheme 1. Synthesis of 1 andnatGa-l.

[0548] Compound 2 enabled the anchoring of the 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10 monoamide (DOTA)-monoamide chelator for metal labeling, and introduction of 6-8044429.601_P18646-02fluoronicotinamide. To evaluate the binding affinity and selectivity of the new agents, inhibition constants (Ki) for FAP, prolyl endopeptidase (PREP), and dipeptidyl dipeptidase 4 (DPPIV) were determined using protein-based competitive binding assays, following our previously developed method. Slania et al., 2021. Compounds 1 (0.13 nM),natGa-l (0.09 nM), andnatIn-l (0.09 nM) displayed high Kivalues for FAP, in the nanomolar range (Table 1 and Table lb), comparable to the known FAP inhibitors FAPI-04 (0.15 nM) and UAMC1110 (0.11 nM) studied in the same assay. The Ki values of those compounds for PREP and DPPIV are in micromolar range, indicating selectivity of the agents for FAP. Jansen et al., 2013; Jansen et al.. 2014; Loktev et al., 2019.Table la. In vitro characterization of representative agents. Molecular weight (MW) and the inhibition constant (Ki) of the agents for FAP, DPPIV, PREP and PSMA are provided. Corresponding units are included in the parenthesis. All agents were studied in the same assay.MW FAP FAP DPPIV PREP(g / mol) IC50 (nM) Ki (nM) IC50(μM) IC50(μM)natGa-l 1057.75 0.44 0.09 4.55 4.37natIn-l 1101.84 0.46 0.09 3.47 2.371 990.44 0.65 0.13 17.87 1.85 FAPI-04 872.93 0.75 0.15 8.81 796 FAPI-46 — — — — —FAPI-74 — — — — —UAMC- 344.32 0.57 0.11 4.43 80.0201110Table lb. In vitro FAP potency and selectivity of 1,natIn-l,natSc-l,natGa-lFAP FAP DPPIV* PREP**Ki IC50 IC50IC50(nM) (nM) (nM) (nM)1 0.13 0.65 17870 1850natIn-l 0.09 0.46 3470 2370natSc-l - - - -8144429.601_P18646-02Table lb. In vitro FAP potency and selectivity of 1,natIn-l,natSc-l,natGa-lFAP FAP DPPIV* PREP**KiIC50IC50IC50(nM) (nM) (nM) (nM)natGa-l (FPyQCP) 0.09 0.44 4550 4370 *dipeptidyl dipeptidase 4 (DPPIV)**prolyl endopeptidase (PREP)

[0549] We docked hFAP-a (PDB ID 1Z68). Aertgeerts et al., 2005. with FAPI-04 and compound 1 using AutoDock following previously reported docking parameters to study in silico binding. Puglioli et al., 2023. That process generated twenty different conformers for each compound ranked based on their respective binding energies. The conformers with the lowest binding energies were selected for further analysis. The docking scores of FAPI-04 (ΔG = -9.6 kcal / mol) and compound 1 (ΔG = -9.2 kcal / mol) were similar, consistent with comparable IC50 values shown in Table 1. Although both 1 (FIG. 2B) and FAPI-04 (FIG. 2C) occupy the catalytic binding pocket of hFAP-α, their binding modes differ significantly. Specifically, the 2-cyanopyrrolidine moiety of 1 is effectively buried within the hydrophobic SI pocket of the protein, composed of Tyr625, Tyr660, Tyr656 and Val705, and is positioned near the catalytic triad residues (Ser624, Asp702, His734). That specific binding mode is further stabilized by hydrogen bonding with Ser348 and hydrophobic interactions involving Gln539 and Val705, facilitated by the fluoropyridine moiety of 1 (FIG. 11B-FIG. 11C). In contrast, the 2-cyanopyrrolidine moiety of FAPI-04 is oriented in the hydrophobic S2 pocket (Phe350, Phe351, Tyr541, Pro544, and Tyr625), distant from the catalytic triad residues (FIG. 2B, FIG. 12A). That orientation of 1 in the FAP binding site is consistent with reported docking studies, particularly the OncoFAP derivatives. Puglioli et al., 2023; Galbiati et al.. 2024.

[0550] Numerous residues participate in hydrophobic interactions with the quinoline moiety of 1 and FAPI-04 (FIG. 11B, FIG. 11C, FIG. 12B, and FIG. 12C). Notably, Phe351 and Phe350 participate in a 7t-stacking interaction with the 7t-cloud (quinoline moiety) of 1, a feature not observed with FAPI-04 (FIG. 11B, FIG. 11C, FIG. 12B, and FIG. 12C). Furthermore, several stabilizing hydrogen bond interactions by different residues surrounding the catalytic triad with 18244429.601_P18646-02and FAPI-04 were observed. The predominant hydrogen bond interactions for FAPI-04 include Arg123, Val540, Tyr541, Ser548, Tyr656, Trp623, Ser624 and His734 (FIG. 11B to FIG. 11C). On the other hand, several non-classical hydrogen bonding contacts between 1 and Tyrl24, Trpl39, Tyr362, Ser548, Trp623, Asn704, and Tyr745 were observed (FIG. 12B to FIG. 12C).

[0551] Radiolabeled analogs,111In-1, and68Ga-l were produced in high radiochemical yields (greater than 95%) and purity (greater than 99%) (FIG. 8, FIG. 9, and FIG. 10).

[0552] All reagents and chemicals are listed in Table 3.Table 3. List of chemicals and solventsManufacturer Reagent NameChem-Impex Na-Boc-Ny-Fmoc-D-2,4-diaminobutyric acid, 0-(7-Azabenzotriazol-l-yl)- N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU).MilliporeSigma Trifluoroacetic acid, Triphosgene, Triethylamine, Piperidine, N, N- Diisopropylethylamine, N, N-Dimethylformamide anhydrous, Dichloromethane anhydrous, 6- fluoronicotinic acid, Triisopropyl silane, Cesium carbonate, 3-(Boc-amino)propyl bromide, ascorbic acid. Kryptofix® 222, 4,7,13.16,21,24-Hexaoxa-l,10-diazabicyclo[8.8.8]hexacosane, Potassium oxalate monohydrate, DMSO anhydrous, Acetonitrile (HPLC grade), Water (HPLC grade).CheMatech DOTA-tris(tBu)ester, NOTA-NHS-ester.Macrocyclics DOTA-NHS -ester.ThermoFisher Scientific Ethyl acetate, Hexane, Acetonitrile anhydrous.

[0553] Compound 1 was further investigated asnatIn / inIn analogs (natIn-l / 111In-1) andnatGa-l / 68Ga-l for in vivo evaluation. Initially,111In-1 and68Ga-1 were assessed in human U87 glioblastoma tumor xenografts in NOD-SCID mice for pharmacokinetics at 1 h (FIG. 11). Tissue biodistribution data [percentage of injected dose per gram (%ID / g) of tissue] of111In-1 are presented in FIG. 3B and Table 4a and Table 4b. High tumor uptake was demonstrated by111In-1, 18.66 ± 6.73 %ID / g, and low accumulation in blood, 4.23 ± 0.47%ID / g, and kidneys, 2.66 ± 0.11%ID / g at 1 h. Most healthy tissues, including heart, lung, liver, muscle, and kidneys, showed low uptake and were in the range of that of blood (2-3 % ID / g). Corresponding tumor-to-tissue8344429.601_P18646-02ratios were greater than three, except for pancreas and bone, known FAP expression sites. Roberts et al., 2013.Table 4a. Tissue biodistribution data of111In-l in U87 xenografts bearing male NOD / SCID mice (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tissue AverageBlood 4.23 ± 0.47Heart 1.69 ± 0.08Lung 3.41 ± 0.15Liver 3.41 + 0.12Stomach 1.55 ± 0.12Pancreas 5.84 ± 0.75Spleen 1.38 ± 0.09Fat 0.63 ± 0.08Kidney 2.66 ± 0.11Muscle 2.53 ± 0.14Small Intestine 3.84 ± 0.60Salivary Gland 9.42 ± 0.63Lacrimal Gland 5.57 ± 0.91Bladder 6.78 ± 2.45Bone 6.45 ± 0.39Tumor (U87) 18.66 ± 6.73Table 4b. Tumor-to-organ ratios of111In-1 in U87 xenografts bearing male NOD / SCID mice (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 1 hTumor-to-Blood 4.35 ± 1.27Tumor-to-Heart 11.09 ± 4.018444429.601_P18646-02Table 4b. Tumor-to-organ ratios of111In-l in U87 xenografts bearing male NOD / SCID mice (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 1 hTumor- to-Lung 5.49 ± 2.01Tumor-to-Liver 5.49 ± 2.06Tumor-to-Stomach 11.86 ± 3.44Tumor-to-Pancreas 3.24 ± 1.31Tumor-to-Spleen 13.58 ± 5.22Tumor-to-Fat 30.53 ± 13.95Tumor-to-Kidney 7.09 ± 2.81Tumor- to-Muscle 7.47 ± 2.97Tumor-to-Small Intestine 4.98 ± 2.19Tumor-to-Salivary Gland 2.00 ± 0.77Tumor-to -Lacrimal Gland 3.43 ± 1.44Tumor-to-Bladder 3.35 ± 2.23Tumor-to-Bone 2.88 ± 0.99

[0554] Biodistribution data of68Ga-l are presented in FIG. 11C and FIG. 11D and Table 5a and Table 5b. The average tumor uptake of68Ga-l at 1 h was higher than111In-l. No significant difference, however, was noted (P > 0.05). The average tumor uptake of68Ga-l remained high up to 2 h, with 20.59 ± 3.30 %ID / g at 0.5 h, 21.32 ± 5.50 %ID / g at 1 h, and 19.96 ± 0.47 at 2 h. Notably, blood uptake of68Ga-l at 1 h (2.73 ± 0.46 %ID / g) was relatively lower than for111In-1, resulting in higher tumor-to-blood ratios (6.38 ± 1.03 at 30 min, 7.91 ± 2.02 at 1 h and 8.10 ± 0.33 at 2 h). Most tumor-to-healthy tissue ratios were significantly higher (P < 0.05) for68Ga-l compared to111In-1. A competitive FAP blocking study was conducted at 1 h post-injection to determine binding specificity using FAPI-04 (500 nmol / kg or approximately 30 nmol / mouse), and showed a significant decrease (P < 0.001) in uptake in the U87 tumors (0.42 ± 0.07 %ID / g), blood (0.35 ± 0.02 %ID / g), and most tissues, except for the kidneys, consistent with kidney being the primary route of clearance of the agent. Those preclinical results motivated us to synthesize the8544429.601_P18646-02corresponding18F-labeled radiotracers to investigate their feasibility for imaging FAP expression in experimental models.Table 5a. Tissue biodistribution data of68Ga-l in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tissue 30 min 1 h 1 h block 2 hBlood 3.23 ± 0.09 2.73 ± 0.46 0.35 ± 0.02 2.47 ± 0.15 Heart 1.11 ± 0.03 0.86 ± 0.18 0.14 ± 0.01 0.69 ± 0.08 Lung 2.37 + 0.17 1.92 ± 0.21 0.37 ± 0.04 1.71 ± 0.07 Liver 1.13 ± 0.16 0.83 ± 0.12 0.26 ± 0.02 1.04 ± 0.05 Stomach 1.57 ± 0.27 1.16 ± 0.22 0.27 ± 0.12 1.01 ± 0.14 Pancreas 2.75 ± 0.24 1.82 ± 0.23 0.13 ± 0.03 1.51 ± 0.29 Spleen 0.80 ± 0.09 0.80 ± 0.29 0.20 ± 0.07 0.85 ± 0.11 Fat 0.68 ± 0.41 0.41 ± 0.24 0.47 ± 0.64 0.43 ± 0.19 Kidney 3.75 ± 0.15 2.77 ± 0.51 2.01 ± 0.13 2.21 ± 0.11 Muscle 2.31 ± 0.21 1.74 ± 0.24 0.12 ± 0.03 1.32 ± 0.25 Small Intestine 2.14 ± 0.35 1.90 ± 0.45 0.29 ± 0.12 1.59 ± 0.37 Salivary Gland 6.98 ± 0.39 4.51 ± 0.74 0.19 ± 0.03 2.62 ± 0.21 Lacrimal Gland 4.91 ± 0.32 3.61 ± 0.78 0.25 ± 0.05 2.20 ± 0.32 Bladder 28.41 ± 32.04 11.10 ± 8.12 14.17 ± 4.49 8.28 ± 7.11 Bone 5.35 ± 0.70 5.11 ± 0.59 0.24 ± 0.16 3.64 ± 0.49 Tumor (U87) 20.59 ± 3.30 21.32 ± 5.50 0.42 ± 0.07 19.96 ± 0.47

[0555] Table 5b. Tumor-to-organ ratios of68Ga-l in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 30 min 1 h 2 hTumor-to-Blood 6.38 ± 1.03 7.91 ± 2.02 8.10 ± 0.33 Tumor- to-Heart 18.47 ± 2.66 26.02 ± 10.09 29.34 ± 4.08 Tumor-to-Lung 8.70 ± 1.24 11.17 ± 3.03 11.70 ± 0.348644429.601_P18646-02Table 5b. Tumor-to-organ ratios of68Ga-l in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 30 min 1 h 2 hTumor- to-Li ver 18.20 ± 1.90 25.37 ± 3.48 19.25 ± 1.00 Tumor-to-Stomach 13.29 ± 2.68 19.24 ± 7.70 20.15 ± 3.06 Tumor- to-Pancreas 7.44 ± 0.62 11.99 ± 3.86 13.65 ± 2.72 Tumor-to-Spleen 25.82 ± 3.34 30.38 ± 16.60 23.91 ± 3.57 Tumor-to-Fat 36.24 + 13.17 105.72 ± 130.03 53.38 ± 23.26 Tumor-to-Kidney 5.50 + 0.87 7.90 ± 2.37 9.04 ± 0.35 Tumor-to-Muscle 8.91 + 0.97 12.75 ± 4.89 15.57 ± 3.23 Tumor- to-Small 9.76 ± 1.71 11.58 ± 3.38 13.09 ± 2.97 IntestineTumor-to-Salivary 2.94 ± 0.32 4.90 ± 1.73 7.64 ± 0.47GlandTumor-to-Lacrimal 4.19 + 0.54 6.19 ± 2.24 9.23 ± 1.41 GlandTumor-to-Bladder 2.15 + 2.49 3.04 ± 2.28 3.47 ± 1.66 Tumor-to-Bone 3.85 ± 0.33 4.19 ± 1.00 5.57 ± 0.80

[0556] Syntheses of the18F-labeled analogs are outlined in FIG. 8A. We began by synthesizing the trimethylammonium precursor, 3, in high yield by conjugating 6-trimethylammonium nicotinic acid tetrafluorophenyl ester to 2 (Scheme 2).(i). DIPEA, DMF, RT, 1 h, 89% (ii). TFA / H2O / TIPS 4 h, RT, 81%o Scheme 2. Synthesis of precursor 3.8744429.601_P18646-02

[0557] The radiosynthesis involved a one-pot Kryptofix 222-assisted nucleophilic reaction of precursor 3 with18F-fluoride followed by metal chelation with Ga(NO3)3or InCl3in acetate buffer (pH 5). Radiochemical yields and purities of the agents are listed in Table 6.Table 6. HPLC Methods for18F-natIn-l and18F-FPyQCP (18F-natGa-l)Tracer Prep HPLC Analytical HPLC Radiochemica Radiochemica Specific codes 1 Yield (RCY) 1 Purity (RCP, Activity HPLC %) (SA. (actual), % Ci / mmol )[18F]1 CH3CN / H2O / TF CH3CN / H2O / TF 50-78 (15-20) >99.9 2500- A 150:850:1 A 150:850:1 5400 Flow 8 ml / min Flow 3 ml / mintR = 23 min tR = 6.1 min18F- CH3CN / H2O / TF CH3CN / H2O / TF 85 (20-22) >99.9 2000-natGa-l A 147:853:1 A 150:850:1 4000 (18F- Flow 7 ml / min Flow 2 ml / minFPyQCP tR = 15 min tR = 4.4 min 55- )18F-111In- CH3CN / H2O / TF CH3CN / H2O / TF 38 (9) >99.9 2550 1 A A160:840:1 160:840:1Flow 8 ml / min Flow 3 ml / mintR = 16.5 min tR = 4.7 min18F-FAPI-74

[0558] The decay-corrected radiochemical yields of18F-1,18F-natIn-l, and18F-natGa-l (called18F-FPyQCP. hereafter) ranged from approximately 25% to 30% based on starting18F-fluoride with absolute yields of 90-180 mCi (3,330-6,660 MBq) after purification by high-performance liquid chromatography (HPLC) (Table 6). Starting from 400-700 mCi (14,800 - 25,900 MBq) of18F-8844429.601_P18646-02fluoride, the specific radioactivity of18F-FPyQCP ranged from 2,500-5400 Ci / mmol (92,500-199,800 GBq / mmol) with radiochemical purity greater than 95%. The identity of the radiolabeled compounds was confirmed by co-injection and co-elution on the HPLC of each radio-fluorinated tracer with the corresponding reference compounds. The18F-labeled analogs proved stable in saline with 7% ethanol for at least 2 h after radio synthesis (FIG. 13, FIG. 14, and FIG. 15).

[0559] 18F-FPyQCP and18F-natIn-l displayed higher tumor uptake and lower blood and normal tissue uptake than18F-1

[0560] The structures of the18F-labeled agents and selected biodistribution data are presented in FIG. 12. As shown in FIG. 12A-FIG. 12B and Table 7a and Table 7b,18F-1, displayed high blood uptake, 16.05 ± 3.22 %ID / g at 1 h after injection along with relatively lower tumor uptake, 7.53 ± 0.34 %ID / g, and higher normal tissue uptake than the radiometal-labeled analogs. Consequently, all tumor-to-normal tissue uptake values were found to be < 1.0. Next, we investigated the non-radiometalated analogs,18F-natIn-l (FIG. 4C, Table 8a and Table 8b) and18F-FPyQCP (FIG. 4D, Table 9a and Table 9b) in tissue biodistribution studies in U87 tumor-bearing NOD-SCID mice. Both agents demonstrated low blood uptake of 2.99 ± 0.43%ID / g (18F-natIn-l) and 3.06 ± 0.14 %ID / g (18F-FPyQCP), as shown in FIG. 4E and FIG. 4F, respectively. The tumor uptake was 13.73 ± 2.68 %ID / g and 25.91 ± 1.66 %ID / g for18F-natIn-l and18F-FPyQCP, respectively. Those tumor uptake values are significantly higher than18F-1. A blocking study with 30 nmol FAPI-04 per mouse (500 nmol / kg) resulted in a significant lowering of tumor uptake (to < 1 %ID / g, P < 0.001), confirming the specificity of these agents. All normal tissues exhibited low uptake, as observed for111In-l and68Ga-l analogs.Table 7a. Tissue biodistribution data of18F-1 in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: 0.74 MBq (20 pCi) / mouse.Tissue 1 h 1 h-blockBlood 16.05 ± 3.22 3.34 ± 2.67Heart 6.95 ± 0.94 0.64 ± 0.61Lung 8.28 ± 1.22 2.32 ± 1.74Liver 6.42 ± 1.34 3.40 ± 3.20Stomach 2.57 ± 0.64 1.09 ± 0.878944429.601_P18646-02Table 7a. Tissue biodistribution data of18F-1 in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: 0.74 MBq (20 pCi) / mouse.Tissue 1 h 1 h-blockPancreas 11.14 ± 0.90 0.73 ± 0.47Spleen 2.74 + 0.16 0.78 ± 0.58Fat 0.44 ± 0.08 0.19 ± 0.04Kidney 13.55 + 4.20 1.92 ± 0.93Muscle 2.44 + 0.12 0.36 ± 0.22Small Intestine 12.12 + 2.63 7.46 ± 8.94Salivary Gland 4.60 + 1.00 0.52 ± 0.61Lacrimal Gland 4.52 ± 0.29 0.63 ± 0.68Bladder 3.57 ± 2.09 8.63 ± 6.03Bone 3.73 ± 0.71 0.63 ± 0.34Tumor (U87) 7.53 ± 0.34 1.38 ± 1.04Table 7b. Tumor-to-organ ratios of18F-1 in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: 0.74 MBq (20 pCi) / mouse.Tumor-to-organ 1 hTumor-to-Blood 0.48 ± 0.08Tumor- to-Heart 1.10 ± 0.12Tumor-to-Lung 0.92 ± 0.11Tumor- to-Li ver 1.20 ± 0.21Tumor-to-Stomach 3.04 ± 0.58Tumor- to-Pancreas 0.68 ± 0.05Tumor-to-Spleen 2.75 ± 0.17Tumor-to-Fat 17.29 ± 2.32Tumor-to-Kidney 0.62 ± 0.28Tumor-to-Muscle 3.09 ± 0.169044429.601_P18646-02Table 7b. Tumor-to-organ ratios of18F-1 in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: 0.74 MBq (20 pCi) / mouse.Tumor-to-organ 1 hTumor-to-Small Intestine 0.64 ± 0.14Tumor-to-Salivary Gland 1.71 ± 0.45Tumor- to-Lacrimal Gland 1.67 ±0.11Tumor-to-Bladder 2.56 ± 1.07Tumor-to-Bone 2.07 ± 0.33Table 8a. Tissue biodistribution data of18F-natIn-l in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tissue 1 h 1 h-blockBlood 2.99 ± 0.43 1.05 ± 0.74Heart 1.17 ± 0.11 0.13 ± 0.01Lung 2.09 ± 0.23 0.47 ± 0.20Liver 2.87 ± 0.27 3.65 ± 0.27Stomach 1.14 ± 0.10 0.29 ± 0.10Pancreas 3.56 ± 0.48 0.40 ± 0.24Spleen 0.96 ± 0.12 0.19 ± 0.05Fat 0.52 ±0.31 0.32 ± 0.24Kidney 2.05 ± 0.23 1.34 ± 0.26Muscle 1.47 ± 0.03 0.84 ± 1.28Small Intestine 3.16 ± 0.46 2.03 ± 0.28Salivary Gland 6.96 ± 1.77 0.25 ± 0.16Lacrimal Gland 4.06 ± 0.75 0.23 ± 0.10Bladder 11.05 ± 10.34 14.15 ± 9.74Bone 4.32 ± 1.09 0.55 ± 0.29Tumor (U87) 13.73 ± 2.68 0.40 ± 0.089144429.601_P18646-02Table 8b. Tumor-to-organ ratios of18F-natIn-l in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 1 hTumor-to-Blood 4.67 ± 1.15Tumor- to-Heart 11.84 ± 2.43Tumor-to-Lung 6.55 ± 0.87Tumor-to-Liver 4.76 ± 0.53Tumor-to-Stomach 12.14 ± 2.52Tumor-to-Pancreas 3.85 ± 0.42Tumor-to-Spleen 14.44 ± 2.53Tumor-to-Fat 32.64 ± 17.21Tumor-to-Kidney 6.79 ± 1.64Tumor-to-Muscle 9.35 ± 1.64Tumor-to-Small Intestine 4.35 ± 0.51Tumor-to-Salivary Gland 2.02 ± 0.39Tumor-to-Lacrimal Gland 3.49 ± 0.99Tumor-to-Bladder 2.70 ± 2.52Tumor-to-Bone 3.36 ± 1.08Table 9a. Tissue biodistribution data ratios of18F-FPyQCP in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tissue 30 min 1 h 1 h-block 2 hBlood 4.25 ± 0.44 3.06 ± 0.14 0.66 ± 0.10 1.87 ± 0.15 Heart 1.50 ± 0.21 1.05 ± 0.08 0.21 ± 0.04 0.71 ± 0.10 Lung 3.06 ± 0.41 2.23 ± 0.17 0.68 ± 0.09 1.37 ± 0.16 Liver 1.50 ± 0.24 1.41 ± 0.13 0.43 ± 0.06 1.21 ± 0.09 Stomach 1.70 ± 0.15 1.07 ± 0.11 0.33 ± 0.04 0.84 ± 0.039244429.601_P18646-02Table 9a. Tissue biodistribution data ratios of18F-FPyQCP in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tissue 30 min 1 h 1 h-block 2 h Pancreas 3.80 ± 0.51 2.68 ± 0.21 0.20 ± 0.00 2.02 ± 0.52 Spleen 1.11 ± 0.22 0.85 ± 0.11 0.25 ± 0.04 0.77 ± 0.21 Fat 0.49 ± 0.09 0.56 ± 0.33 0.10 ± 0.06 0.34 ± 0.13 Kidney 3.42 ± 0.38 2.54 ± 0.17 2.61 ± 0.34 2.19 ± 0.30 Muscle 2.40 ± 0.26 2.12 ± 0.25 0.21 ± 0.16 1.58 ± 0.33 Small Intestine 3.23 ± 0.65 2.59 ± 0.26 0.44 ± 0.06 1.80 ± 0.38 Salivary Gland 7.98 ± 1.01 5.35 ± 0.54 0.29 ± 0.05 3.91 ± 0.59 Lacrimal Gland 5.32 ± 0.88 3.91 ± 0.51 0.31 ± 0.09 3.06 ± 0.44 Bladder 12.32 ± 5.57 8.57 ± 7.35 13.47 ± 13.75 9.57 ± 7.72 Bone 7.07 ± 0.60 6.67 ± 0.84 0.28 ± 0.06 4.90 ± 0.87 U87 22.35 ± 1.88 25.91 ± 1.66 0.54 ± 0.08 23.28 ± 2.05Table 9b. Tumor-to-organ ratios of18F-FPyQCP in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 30 min 1 h 2 hTumor-to-Blood 5.27 ± 0.17 8.48 ± 0.68 12.03 ± 0.30 Tumor- to-Heart 14.99 ±1.49 24.73 ± 2.49 33.13 ± 1.86 Tumor-to-Lung 7.34 ± 0.53 11.63 ± 0.44 17.12 ± 1.33 Tumor-to-Liver 15.10 ± 1.65 18.45 ± 2.11 19.22 ± 0.47 Tumor-to-Stomach 13.20 ± 1.17 24.41 ± 3.25 27.79 ± 2.52 Tumor-to-Pancreas 5.91 ± 0.33 9.71 ± 1.06 11.93 ± 2.26 Tumor-to-Spleen 20.53 ± 3.22 30.61 ± 3.57 32.03 ± 8.98 Tumor-to-Fat 46.59 ± 5.40 60.69 ± 33.14 77.39 ± 26.94 Tumor-to-Kidney 6.56 ± 0.52 10.24 ± 0.81 10.72 ± 0.78 Tumor-to-Muscle 9.36 ± 0.83 12.41 ± 2.14 15.09 ± 2.109344429.601_P18646-02Table 9b. Tumor-to-organ ratios of18F-FPyQCP in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 30 min 1 h 2 hTumor-to-Small 7.09 ± 1.23 10.05 ± 0.76 13.39 ± 3.25 IntestineTumor-to-Salivary 2.81 ± 0.21 4.90 ± 0.73 6.01 ± 0.50 GlandTumor-to-Lacrimal 4.25 ± 0.50 6.71 ± 0.87 7.65 ± 0.44 GlandTumor-to-Bladder 2.26 ± 1.34 5.92 ± 5.94 4.26 ± 3.34 Tumor-to-Bone 3.17 ± 0.26 3.93 ± 0.55 4.83 ± 0.65Table 9c. Tissue biodistribution data and tumor-to-organ ratios of [18F]natSc-l in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 30 pCi (1.1 MBq) / mouse.Tissue 1 h 1 h-blockBlood 2.91 ± 0.03 0.06 ± 0.01Heart 1.51 ± 0.07 0.04 ± 0.01Lung 1.71 ± 0.20 0.20 ± 0.04Liver 5.82 ± 0.04 6.20 ± 0.97Stomach 1.22 ± 0.13 0.21 ± 0.09Pancreas 5.34 ± 1.32 0.08 ± 0.04Spleen 0.88 ± 0.13 0.21 ± 0.20Fat 0.51 ± 0.09 0.07 ± 0.03Kidney 1.60 ± 0.09 1.20 ± 0.13Muscle 1.75 ± 0.23 0.07 ± 0.05Small Intestine 2.39 ± 0.52 1.74 ± 1.06Salivary Gland 6.82 ± 0.07 0.05 ± 0.01Bladder 1.73 ± 0.74 0.55 ± 0.399444429.601_P18646-02Table 9c. Tissue biodistribution data and tumor-to-organ ratios of [18F]natSc-l in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 30 pCi (1.1 MBq) / mouse.Tissue 1 h 1 h-blockBone 3.67 ± 0.41 0.43 ± 0.10U87 7.38 ± 0.52 0.13 ± 0.03Table 9d. Tissue biodistribution data and tumor-to-organ ratios of [18F]natSc-l in male NOD / SCID mice bearing U87 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 30 pCi (1.1 MBq) / mouse.Tumor-to-organ 1 hTumor-to-Blood 2.54Tumor-to-Heart 4.89Tumor-to-Lung 4.32Tumor-to-Liver 1.27Tumor-to-Stomach 6.05Tumor-to-Pancreas 1.38Tumor-to-Spleen 8.38Tumor-to-Fat 14.47Tumor-to-Kidney 4.61Tumor-to-Muscle 4.22Tumor-to-Small Intestine 3.09Tumor-to-Salivary Gland 1.08Tumor-to-Bladder 4.27Tumor-to-Bone 2.01

[0561] A biodistribution study of18F-FPyQCP was also conducted up to 2 h in U87 tumor-bearing mice. Blood uptake went down to 1.87 ± 0.15 %ID / g at 2 h while tumor uptake remained in the same range (23.28 ± 2.05 %ID / g), resulting in a high tumor-to-normal organ ratio, for example, tumor-to-blood, 5.27 ± 0.17 at 0.5 h, 8.48 ± 0.68 at 1 h and 12.03 ± 0.30 at 2 h, respectively. Similarly, high tumor-to-liver (19.22 ± 0.47), tumor-to-kidney (10.72 ± 0.78) and tumor-to-muscle 9544429.601_P18646-02ratios (15.09 ± 2.10) indicated improved clearance of the agent at 2 h after injection. Overall, the tumor uptake and in vivo pharmacokinetics of18F-natIn-l and18F- FPyQCP were nearly identical to their corresponding radiometal analogs, and18F-FPyQCP displayed relatively lower nonspecific tissue uptake and blood uptake than18F-1 (P < 0.01). Furthermore, tumor uptake of18F-FPyQCP was significantly higher (P < 0.001) than18F-natIn-l and18F-FPyQCP.

[0562] 18F-FPyQCP demonstrated increased tumor uptake relative to18F-AlF-FAPI-74 in experimental murine models with variable FAP expression

[0563] Given the superior pharmacokinetics and tumor targeting of18F-FPyQCP compared to18F-natIn-l, we next evaluated18F-FPyQCP head-to-head with18F-AlF-FAPI-74 in three commonly used xenografts with variable FAP expression, namely, those derived from human U87, Capan-2, and HT1080-FAP cells. Flow cytometry data confirmed the FAP expression levels as U87 > HT1080-FAP > Capan-2 > HT1080, as shown in FIG. 5A-B.

[0564] Biodistribution data at 1 h are presented in FIG. 5C-E. We found that tumor uptake of18F-FPyQCP (18.21 ± 3.31% ID / g) (FIG. 5C, Table 10a and Table 10b) was greater than 2-fold higher than18F-AlF-FAPI-74 (7.31 ± 3.49 % ID / g) (P < 0.001). Additionally, significantly higher blood (2.2 ± 0.44 %ID / g vs 0.89 ± 0.02 %ID / g), pancreas (3.72 ± 0.73 %ID / g vs. 1.07 ± 0.1 %ID / g), salivary glands (4.19 ± 0.28 %ID / g vs. 1.36 ± 0.13 %ID / g) uptake was observed for18F-FPyQCP vs.18F-AlF-FAPI-74, respectively. These high normal tissue uptake in the pancreas and salivary glands is expected for18F-FPyQCP because of high FAP expression in those tissues. Keane et al., 2014.Table 10a. Tissue biodistribution data ratios of18F-FPyQCP and18F-AlF-FAPI-74 in male Nude mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h postinjection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.18F-FPyQCP18F-AlF-FAPI-74 Tissue I h 1 h-block 1 h 1 h-block Blood 2.2 ± 0.44 0.28 ± 0.14 0.89 ± 0.02 0.36 ± 0.13 Heart 1 ± 0.13 0.13 ± 0.06 0.48 ± 0.05 0.15 ± 0.06 Lung 1.59 ± 0.21 0.4 ± 0.15 0.88 ± 0.06 0.52 ± 0.15 Liver 1.06 ± 0.13 0.36 ± 0.1 1.34 ± 0.08 1.48 ± 0.24 Stomach 0.83 ± 0.16 0.18 ± 0.03 0.45 ± 0.05 0.3 ± 0.139644429.601_P18646-02Table 10a. Tissue biodistribution data ratios of18F-FPyQCP and18F-AlF-FAPI-74 in male Nude mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h postinjection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.18F-FPyQCP18F-AlF-FAPI-74 Tissue I h 1 h-block 1 h 1 h-block Pancreas 3.72 ± 0.73 0.13 ± 0.07 1.07 ± 0.1 0.17 ± 0.06 Spleen 0.65 ± 0.17 0.12 ± 0.08 0.29 ± 0.04 0.18 ± 0.08 Fat 0.52 ± 0.14 0.42 ± 0.7 0.32 ± 0.31 0.17 ± 0.14 Kidney 1.78 ± 0.1 1.78 ± 0.28 1.93 ± 0.17 2.02 ± 0.49 Muscle 1.89 ± 0.33 0.05 ± 0.05 0.69 ± 0.15 0.1 ± 0.02 Femur 3.57 ± 0.19 0.13 ± 0.04 2.5 ± 0.39 0.57 ± 0.13 Small intestine 1.03 ± 0.22 0.22 ± 0.08 0.6 ± 0.05 0.59 ± 0.2 Large intestine 1.02 ± 0.12 0.2 ± 0.1 0.44 ± 0.16 0.25 ± 0.11 Salivary gland 4.19 ± 0.28 0.13 ± 0.07 1.36 ± 0.13 0.14 ± 0.05 Bladder 0.92 ± 0.12 1.38 ± 0.45 9.29 ± 0.28 2.48 ± 1.37 Brain 0.07 ± 0.01 0.01 ± 0.01 0.04 ± 0.01 0.02 ± 0.02 Tumor 18.21 ± 3.31 0.57 ± 3.16 7.31 ± 3.49 0.41 ± 0.07Table 10b. Tumor-to-organ ratios of18F-FPyQCP and18F-AlF-FAPI-74 in male Nude mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 1 h 1 h18F-FPyQCP18F- A1F-FAPI-74 Tumor- to-Blood 8.49 ± 1.77 8.23 ± 0.61Tumor-to-Heart 18.60 ± 4.11 15.46 ± 2.42Tumor- to-Lung 13.62 ± 1.60 8.36 ± 0.60Tumor-to-Liver 17.23 ± 3.28 5.48 ± 0.58Tumor-to-Stomach 22.73 ± 7.75 16.61 ± 2.57Tumor- to-Pancreas 5.14 ± 0.99 6.87 ± 0.96Tumor-to-Spleen 31.21 ± 9.26 25.60 ± 4.579744429.601_P18646-02Table 10b. Tumor-to-organ ratios of18F-FPyQCP and18F-AlF-FAPI-74 in male Nude mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4) at 1 h post-injection. Injection Dose: approximately 20 pCi (0.74 MBq) / mouse.Tumor-to-organ 1 h 1 h18F-FPyQCP18F- A1F-FAPI-74Tumor-to-Fat 36.91 ± 5.11 34.30 ± 15.52Tumor- to-Kidney 9.56 ± 2.59 3.83 ± 0.51Tumor-to-Muscle 10.41 ± 2.10 10.83 ± 1.91Tumor-to-Femur 5.48 ± 1.24 3.00 ± 0.60Tumor-to-Small intestine 17.82 ± 4.85 12.37 ± 1.69Tumor-to-Large intestine 18.40 ± 3.80 20.32 ± 12.62Tumor-to-Salivary gland 4.47 ± 0.63 5.43 ± 0.79Tumor-to-Bladder 19.91 ± 2.43 2.82 ± 3.46Tumor-to-Brain 288.53 ± 56.50 178.80 ±42.77

[0565] In the HT1080-FAP model,18F-FPyQCP (8.14 ± 1.71 %ID / g) demonstrated approximately 1.5-fold higher uptake than18F-AlF-FAPI-74 (5.37 ± 1.65 %ID / g) (FIG. 5D, Table Ila and Table 1 lb). An approximately 2-fold higher tumor uptake of 8.48 ± 1.1 % (18F-FPyQCP) vs. 4.42 ± 0.68 % (18F-AlF-FAPI-74) in Capan-2 models was demonstrated (FIG. 5E, Table 12a and Table 12b). Those findings were further validated using68Ga-l analog in the Capan-2 model (Table 13a and Table 13b). Immunohistochemistry studies confirmed that higher FAP expression was associated with the HT1080-FAP and U87 tumors compared to the Capan-2 model (P <0.001) (FIG. 5F -FIG. 5G). Furthermore, we synthesized18F-FPyQCP (D) by substituting the linker, L-2,4-aminobutyric acid, with D-2,4-aminobutyric acid and evaluated this agent in NOD-SCID mice bearing U87 tumors. The results showed no significant difference between18F-FPyQCP and its isomeric analog18F-FPyQCP (D) in blood and normal tissue uptake (Table 14a and Table 14b), while somewhat lower tumor uptake was noted.9844429.601_P18646-02Table 11a. Tissue biodistribution data of18F-FPyQCP and18F- AlF-FAPI-74 in male Nude mice bearing HT1080-FAP xenografts (Data are % ID / g, expressed)18F-FPyQCP18F- A1F-FAPI-74 Tissue I h 1 h-block 1 h 1 h-block Blood 2.31 ± 0.19 0.89 ± 1.13 0.88 ± 0.04 0.49 ± 0.31 Heart 1.2 ± 0.11 0.2 ± 0.14 0.54 ± 0.04 0.27 ± 0.14 Lung 1.74 ± 0.28 0.45 ± 0.17 0.94 ± 0.08 0.67 ± 0.25 Liver 1.26 ± 0.1 0.42 ± 0.08 1.49 ± 0.1 1.82 ± 0.59 Stomach 0.98 ± 0.07 0.27 ± 0.19 0.49 ± 0.07 0.29 ± 0.13 Pancreas 4.07 ± 0.26 0.2 ± 0.07 1.11 ± 0.15 0.19 ± 0.09 Spleen 0.81 ± 0.27 0.17 ± 0.03 0.51 ± 0.2 0.37 ± 0.2 Fat 0.88 ± 0.17 0.14 ± 0.16 0.7 ± 0.77 0.51 ± 0.86 Kidney 2.25 ± 0.2 1.86 ± 0.32 2.09 ± 0.15 5.43 ± 6.88 Muscle 1.94 ± 0.3 0.11 ± 0.05 0.7 ± 0.1 0.39 ± 0.26 Femur 4.67 ± 1.08 0.2 ± 0.06 2.48 ± 0.31 0.54 ± 0.41 Small intestine 1.33 ± 0.39 0.31 ± 0.19 0.7 ± 0.19 0.64 ± 0.24 Large intestine 1.4 ± 0.18 0.2 ± 0.07 0.58 ± 0.05 0.29 ± 0.16 Salivary gland 4.55 ± 0.69 0.16 ± 0.09 1.31 ± 0.15 0.21 ± 0.15 Bladder 1.78 ± 0.83 0.96 ± 0.25 9.49 ± 5.96 2.87 ± 2.76 Brain 0.08 ± 0.01 0.02 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 Tumor 8.14 ± 1.71 0.66 ± 0.17 5.37 ± 1.65 1.03 ± 0.9Table 11b. Tumor-to-organ ratios of18F-FPyQCP and18F-AlF-FAPI-74 in male Nude mice bearing HT1080-FAP xenografts (Data are % ID / g, expressed)Tumor-to-organ Average ( 1 h)18F-FPyQCP18F- A1F-FAPI-74 Tumor-to-Blood 3.53 ± 0.67 6.02 ± 1.67Tumor-to-Heart 6.76 ± 0.96 10.11 ± 3.91Tumor-to-Lung 4.03 ± 0.73 6.27 ± 0.51Tumor-to-Liver 6.40 ± 0.83 3.62 ± 1.229944429.601_P18646-02Table 11b. Tumor-to-organ ratios of18F-FPyQCP and18F-AlF-FAPI-74 in male Nude mice bearing HT1080-FAP xenografts (Data are % ID / g, expressed)Tumor-to-organ Average ( 1 h)18F-FPyQCP18F- AlF-FAPI-74 Tumor-to-Stomach 8.31 ± 1.56 11.06 ± 3.72Tumor-to-Pancreas 2.01 ± 0.44 4.92 ± 1.64Tumor-to-Spleen 10.36 ± 1.38 12.99 ± 8.62Tumor-to-Fat 9.38 ± 2.05 19.18 ± 19.65Tumor-to-Kidney 3.67 ± 1.06 2.55 ± 0.73Tumor-to-Muscle 4.29 ± 1.20 7.90 ± 2.81Tumor-to-Femur 1.79 ± 0.42 2.13 ± 0.42Tumor-to-Small intestine 6.54 ± 2.23 8.18 ± 3.85Tumor-to-Large intestine 5.96 ± 1.96 9.46 ± 3.45Tumor-to-Salivary gland 1.80 ± 0.28 4.11 ± 1.15Tumor-to-Bladder 5.01 ± 1.46 0.86 ± 0.64Tumor-to-Brain 103.41 ± 25.83 120.97 ± 54.09as mean ± SD) (n = 4 or 5) at 1 h post-injection. Injection Dose: ~20 pCi (0.74 MBq) / mouse.Table 12a. Tissue biodistribution data of18F-FPyQCP and18F- A1F-FAPI-74 in male Nude mice bearing Capan-2 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.18F-FPyQCP18F- A1F-FAPI-74 Tissue I h 1 h-block 1 h 1 h-block Blood 1.93 ± 0.1 0.33 ± 0.15 0.96 ± 0.13 0.22 ± 0.11 Heart 1.06 ± 0.08 0.18 ± 0.05 0.54 ± 0.06 0.11 ± 0.03 Lung 1.31 ± 0.2 0.45 ± 0.11 0.9 ± 0.11 0.35 ± 0.08 Liver 1.03 ± 0.09 0.41 ± 0.06 1.66 ± 0.33 1.4 ± 0.22 Stomach 0.85 ± 0.13 0.23 ± 0.09 0.46 ± 0.06 0.16 ± 0.04 Pancreas 3.65 ± 0.35 0.16 ± 0.02 1.03 ± 0.12 0.07 ± 0.03 Spleen 0.85 ± 0.21 0.2 ± 0.06 0.4 ± 0.09 0.14 ± 0.0210044429.601_P18646-02Fat 0.92 ± 0.61 0.17 + 0.07 1.46 ± 2.55 0.06 ± 0.05 Kidney 1.77 ± 0.09 2.16 ± 0.45 2.07 ± 0.13 1.55 ± 0.26 Muscle 2.35 ± 0.66 0.12 ± 0.05 0.69 ± 0.17 0.13 ± 0.1 Femur 3.99 + 0.71 0.18 ± 0.06 2.31 ± 0.26 0.72 ± 0.35 Small intestine 1.05 + 0.23 0.24 ± 0.07 0.81 ± 0.2 0.64 ± 0.14 Large intestine 0.95 + 0.08 0.23 ± 0.04 0.5 ± 0.04 0.16 ± 0.08 Salivary gland 4.36 ± 0.65 0.2 ± 0.09 1.41 ± 0.11 0.1 ± 0.03 Bladder 1.21 ± 0.48 1.12 ± 1.11 6.24 ± 6.03 1.46 ± 1.97 Brain 0.08 ± 0.03 0.03 ± 0 0.04 ± 0 0.02 ± 0.01 Tumor 8.48 + 1.1 0.73 ± 0.14 4.42 ± 0.68 0.46 ± 0.06Table 12b. Tumor-to-organ ratios of18F-FPyQCP and18F- A1F-FAPI-74 in male Nude mice bearing Capan-2 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.Tumor-to-organ 1 h Ih18F-FPyQCP18F- A1F-F API-74 Tumor- to-Blood 4.41 ± 0.73 4.69 ± 0.93Tumor-to-Heart 8.03 ± 1.36 8.32 ± 1.68Tumor- to-Lung 6.97 ± 1.13 4.23 ± 0.48Tumor-to-Liver 8.30 ± 1.61 2.71 ± 0.53Tumor-to-Stomach 10.15 ± 1.80 9.65 ± 0.99Tumor-to-Pancreas 2.34 ± 0.37 4.35 ± 0.98Tumor-to-Spleen 10.73 ± 3.62 11.39 ± 3.09Tumor-to-Fat 12.08 ± 5.73 12.80 ± 8.50Tumor-to-Kidney 4.81 ± 0.65 2.14 ± 0.25Tumor- to-Muscle 3.92 ± 1.39 6.78 ± 2.06Tumor-to-Femur 2.20 ± 0.57 1.94 ± 0.42Tumor-to-Small intestine 8.50 ± 2.54 5.77 ± 1.86Tumor-to-Large intestine 8.91 ± 1.15 8.78 ± 1.24Tumor-to-Salivary gland 1.98 ± 0.38 3.13 ± 0.3310144429.601_P18646-02Table 12b. Tumor-to-organ ratios of18F-FPyQCP and18F- AlF-FAPI-74 in male Nude mice bearing Capan-2 xenografts, (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.Tumor-to-organ 1 h Ih18F-FPyQCP18F- A1F-FAPI-74 Tumor-to-Bladder 7.77 ± 2.82 1.75 ± 1.83Tumor-to-Brain 116.22 ± 41.46 121.35 ± 12.02Table 13a. Tissue biodistribution data of68Ga-l in male Nude mice bearing HT-1080FAP xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.Tissue AverageBlood 3.29 ± 0.75Heart 1.41 + 0.30Lung 2.17 + 0.48Liver 1.27 + 0.27Stomach 1.18 ± 0.30Pancreas 1.50 ± 0.33Spleen 2.38 ± 0.99Fat 1.22 ± 0.76Kidney 3.27 ± 0.74Muscle 1.89 ± 0.46Small Intestine 1.41 ± 0.15Salivary Gland 5.64 ± 1.84Lacrimal Gland 4.91 ± 2.02Bladder 9.28 ± 5.69Bone 6.93 ± 1.33Tumor 7.38 ± 2.1010244429.601_P18646-02Table 13b. Tumor-to-organ ratios of68Ga-l in male Nude mice bearing HT-1080FAP xenografts (Data are % ID / g, expressed as mean ± SD) (n = 4). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.Tumor-to-organ AverageTumor- to-Blood 2.23 ± 0.32Tumor-to-Heart 5.35 ± 1.67Tumor- to-Lung 3.47 ± 0.99Tumor-to-Liver 5.77 ± 0.80Tumor-to-Stomach 6.56 ± 2.43Tumor- to-Pancreas 5.02 ± 1.45Tumor-to-Spleen 3.38 ± 1.14Tumor- to-Fat 7.17 ± 3.01Tumor-to-Kidney 2.28 ± 0.53Tumor- to-Muscle 3.90 ± 0.67Tumor-to-Small Intestine 5.24 ± 1.31Tumor-to-Salivary Gland 1.33 ± 0.22Tumor- to-Lacrimal Gland 1.76 ± 1.08Tumor-to-Bladder 0.92 ± 0.29Tumor-to-Bone 1.05 ± 0.12Table 14a. Tissue biodistribution data and tumor-to-organ ratios of18F-FPyQCP (D) in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.18F- FPyQCP (D)Tissue 1 h 1 h-blockBlood 4.04 ± 3.10 0.17 ± 0.06Heart 1.16 ± 0.16 0.10 ± 0.04Lung 2.12 ± 0.19 0.30 ± 0.06Liver 1.18 ± 0.13 0.30 ± 0.08Stomach 1.20 ± 0.28 0.14 ± 0.0310344429.601_P18646-02Table 14a. Tissue biodistribution data and tumor-to-organ ratios of18F-FPyQCP (D) in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.18F- FPyQCP (D)Pancreas 3.74 ± 1.60 0.10 ± 0.02Spleen 1.14 + 0.14 0.11 ± 0.06Fat 0.51 ± 0.14 0.08 ± 0.10Kidney 7.15 ± 10.44 1.93 ± 0.22Muscle 3.53 ± 4.49 0.05 ± 0.01Femur 3.34 + 0.70 0.13 ± 0.05Small intestine 1.85 ± 0.39 0.30 ± 0.32Large intestine 1.48 ± 0.25 0.12 ± 0.03Salivary gland 3.54 ± 1.80 0.14 ± 0.05Bladder 1.06 ± 0.28 1.48 ± 2.61Brain 0.09 ± 0.01 0.09 ± 0.15Tumor 11.91 ± 1.58 0.91 ± 0.87Table 14b. Tumor-to-organ ratios of18F-FPyQCP (D) in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.Tumor-to-organ AverageTumor- to-Blood 0.36 ± 0.33Tumor-to-Heart 0.10 ± 0.01Tumor- to-Lung 0.18 ± 0.01Tumor-to-Liver 0.10 ± 0.01Tumor-to-Stomach 0.10 ± 0.03Tumor-to-Pancreas 0.31 ± 0.11Tumor-to-Spleen 0.10 ± 0.01Tumor-to-Fat 0.04 ± 0.01Tumor-to-Kidney 0.67 ± 1.0610444429.601_P18646-02Table 14b. Tumor-to-organ ratios of18F-FPyQCP (D) in male NOD / SCID mice bearing U87 xenografts (Data are % ID / g, expressed as mean ± SD) (n = 5). Injection Dose: approximately 20 pCi (0.74 MBq) / mouse and 1 h post injection.Tumor-to-organ AverageTumor- to-Muscle 0.33 ± 0.46Tumor-to-Femur 0.28 + 0.02Tumor-to-Small intestine 0.16 ± 0.03Tumor-to-Large intestine 0.12 + 0.02Tumor-to-Salivary gland 0.29 + 0.13Tumor-to-Bladder 0.09 + 0.01Tumor-to-Brain 0.01 + 0.00

[0566] Overall, these data suggest that18F-FPyQCP may provide improved targeting to FAP+ tumors relative to18F-AlF-FAPI-74. In addition, favorable tumor-to-background ratios were achieved with18F-FPyQCP, as summarized in FIG. 5H. Although tumor-to-blood and tumor-to-muscle ratios are nearly similar (P > 0.05), significantly higher tumor-to-liver and tumor-to-kidney ratios were noted for18F-FPyQCP (P < 0.001) compared to18F-AlF-FAPI-74 suggesting that18F-FPyQCP might offer comparable or improved PET imaging in FAP+ tumors.

[0567] Dynamic PET scans of18F-FPyQCP were conducted in U87 tumor-bearing mice (n = 2), as presented in FIG. 6 A - FIG. 6B. An early peak in the tumor was observed, followed by slightly increased uptake and retention over the duration of the PET scan. Initial high uptake was observed in the heart, which decreased after 30 min. The kidney uptake was high initially and displayed rapid clearance, reducing to the baseline by 90 min. Remarkably low normal tissue uptake was observed in liver and abdominal tissues, consistent with static imaging. Those imaging data and tumor uptake values aligned well with the ex vivo tissue biodistribution data.

[0568] Static PET / MR imaging data of18F-FPyQCP and PET / MR imaging of18F-FPyQCP and18F-FAP-74 are presented side-by-side in FIG. 6C, showcasing imaging at 30 min, 60 min, and 120 min post-injection in U87 tumor-bearing mice. Both radiotracers exhibited high uptake in tumor and low background tissue uptake except for the bladder, although there are some differences. The mouse injected with18F-FPyQCP showed a relatively higher salivary gland and bone marrow than the mouse injected with18F-FAP-74. Conversely, high uptake was observed in10544429.601_P18646-02the liver, gallbladder, and intestines in mice injected with18F-FAP-74. Notably, radiotracer uptake continued to increase in those tissues at 60 min and 120 min in mouse injected in18F-A1F-FAPI-74, indicating differences in metabolic and clearance pathways that were not noticeable from the ex vivo tissue biodistribution studies.18F-FPyQCP was cleared from normal tissues more rapidly between 30- and 120-min post-injection, with minimal liver, gallbladder or intestinal uptake. The data suggest that renal clearance is the primary route because of the high radiotracer concentration in the bladder for both agents, consistent with other FAP-based small molecule agents.18F-FPyQCP demonstrated remarkably low hepatobiliary clearance. Although the stability in PBS for both agents is similar (FIG. 18),18F-FPyQCP showed superior in vivo pharmacokinetics compared to18F-AlF-FAP-74, as we hypothesized. Additionally,18F-FPyQCP demonstrated similar tumor uptake and pharmacokinetics to68Ga-l (P > 1).

[0569] Consistent with previous reports, high radiotracer accumulation also was detected in the skull and bone joints, relatively higher for18F-FPyQCP than18F-AlF-FAPI-74. Receptor-blocking studies revealed significantly reduced bone-joint uptake for both compounds. A head-to-head FAP blocking study (30 nmol / mouse) showed relatively higher hepatobiliary clearance of18F-A1F-FAP-74 compared to18F-FPyQCP (FIG. 6D). That observation is consistent with reported studies, which suggested that binding of these FAP-based agents is owing to FAP expression in the synovial fluid. Breznik et al., 2017; Tanc et al., 2024; Laverman et al., 2015. Minimal bone uptake, however, is noted in human PET imaging, indicating that high bone-joint uptake in mice might not be extrapolated to patients.

[0570] 18F-FPyQCP demonstrates low non-specific tissue uptake in a healthy male baboon compared toI8F-AlF-FAPI-74

[0571] The internal radiation dosimetry in patients was estimated based on the PET imaging in a non-human primate (Papio anubis), and selected tissue-absorbed data are presented in Table 15. The PET imaging data of both agents are provided in FIG. 7. The highest absorbed dose for18F-FPyQCP was in the urinary bladder wall (4.55E-02 mSv / MBq), followed by the prostate 2.36E-02 mSv / MBq, kidneys (2.12E-02 mSv / MBq), pancreas (1.12E-02 mSv / MBq) and red marrow (1.01E-02 mSv / MBq). These absorbed doses are much lower than18F-A1F-NOTA-FAPI. Witek et al., 2024.10644429.601_P18646-02Table 15. Organ-Absorbed Dose and Effective Dose for18F-FPyQCPand18F-AlF-FAPI-74.18F-FPyQCP18F-AlF-FAPI-74Tissue mSv / MBq mSv / MBqAdipose tissue 9.24E-03 1.30E-02Adrenals 1.23E-02 5.00E-03Alveolar-interstitial 5.73E-03 5.00E-03Bronchioles secretary cells 5.73E-03 2.00E-03Brain 2.38E-03 7.00E-03Breast 7.37E-03 4.00E-03Bronchi basal cells 4.41E-03 4.00E-03Bronchi secretary cells 4.41E-03 8.00E-03Endosteal cells 8.90E-03 4.00E-03ET1 basal cells 3.90E-03 5.00E-03ET2 basal cells 5.45E-03 6.00E-03Lens of the eye 5.71E-03 2.20E-02Gall bladder wall 1.14E-02 7.00E-03Heart wall 7.58E-03 2.10E-02Kidneys 2.12E-02 9.00E-03Left colon stem cell layer 1.02E-02 1.00E-02Liver 7.79E-03 8.00E-03Extrathroacic lymph nodes 8.92E-03 1.10E-02Systemic lymph nodes 1.14E-02 8.00E-03Thoracic lymph nodes 8.72E-03 5.00E-03Muscle 8.37E-03 8.00E-03Oral Mucosa 7.91E-03 8.00E-03Oesophagus 8.51E-03 6.00E-03Pituitary gland 6.40E-03 1.50E-02Pancreas 1.12E-02 1.90E-02Prostate 2.36E-02 9.00E-0310744429.601_P18646-02Table 15. Organ-Absorbed Dose and Effective Dose for18F-FPyQCP and18F-AlF-FAPI-74.18F-FPyQCP18F-AlF-FAPI-74Tissue mSv / MBq mSv / MBqRed marrow 1.01E-02 1.00E-02Right colon stem cell layer 1.06E-02 1.40E-02Rectosigmoid Colon stem1.68E-02cell layer 7.00E-03Salivary glands 7.61E-03 1.10E-02Small intestine stem cell1.22E-02layer 7.00E-03Skin 6.95E-03 7.00E-03Spleen 7.67E-03 9.00E-03Stomach stem cell layer 9.17E-03 9.00E-03Testes 9.84E-03 8.00E-03Thymus 8.47E-03 8.00E-03Thyroid 8.56E-03 5.00E-03Tongue 5.58E-03 8.00E-03Tonsils 8.08E-03 3.60E-02Urinary bladder wall 4.55E-02 1.30E-02Ureters 1.47E-02 1.30E-02Effective dose coefficient 1.06E-02 1.00E-02

[0572] For18F-AlF-FAPI-74, the organs with the highest radiation-absorbed doses included kidneys (3.60E-02 mSv / MBq), followed by skin (2.20E-02 mSv / MBq), heart wall (2.10E-02 mSv / MBq) and testes (1.40E-02 mSv / MBq). Other tissues with notably high absorbed doses were adipose tissue, urinary bladder, and prostate (1.30E-02 mSv / MBq). Liver and salivary gland absorbed doses were similar for both agents. The estimated effective dose was 0.10E-02 mSv / MBq for18F-FAPI-74 and 0.11E-02 mSv / MBq for18F-FPyQCP, respectively. These values are in a similar range to other18F-based radiotracers. For example, 144 different18F-labeled radiotracers were found to have a mean effective dose of 2.05E-2 mSv / MBq. Zanotti-Fregonara et al., 2021.10844429.601_P18646-02Those data support that18F-FPyQCP PET yields an acceptable radiation dose to humans, suggesting its suitability for patient studies.

[0573] Discussion

[0574] We sought to extend our previous work leveraging the 2-cyanopyrrolidine aminoquinoline scaffold for radionuclide-based imaging of FAP to PET. Slania et al., 2021. Accordingly, we developed a new series of PET imaging agents with a 6-fluoronicotinamide moiety using traditional ways of introducing18F that contain a covalent C-F bond. Another impetus for introducing a 6-fluoronicioinamide moiety was that an industrial- scale synthesis would be possible using a single-step, semi-automated method for clinical translation. Furthermore, the presently disclosed subject matter demonstrates the effect of the presence or absence of a radiometal chelated within the DOTA-monoamide chelating moiety on overall radiotracer pharmacokinetics. Those strategies have not been pursued in FAP-based PET imaging previously.

[0575] Last, we compared18F-FPyQCP to18F-AlF-FAPI-74, among the best-performing of all FAP-targeted imaging agents that have been translated to date. Archibald and Allott, 2021; Xia et al., 2020. We synthesized18F-1,18F-natIn-1, and18F-FPyQCP and compared them to18F-A1F-FAPI-74. Without wishing to be bound to any one particular theory, it was thought that a metal ion in the chelator might provide slightly more hydrophilic if not charged compounds, with consequently lower protein binding and improved pharmacokinetics. Xu et al., 2021.

[0576] To this end, the higher blood uptake and healthy tissue retention observed with18F-natIn-l compared to18F-FPyQCP is likely linked to differences in their overall charge and the ionic radii of the metals involved. Gallium-68, with a smaller ionic radius compared to indium- 111, tends to result in a pendant acetate group from the DOTA chelator, as shown by the X-ray crystal structure of68Ga-DOTA complexes in its six-fold coordination in pseudo-octahedral arrangements. Xie et al., 2021; Viola et al., 2006; Banerjee et al., 2010. That structural feature is believed to improve tumor targeting and fast pharmacokinetics of18F-FPyQCP. Such68Ga-based receptor-targeted imaging compared to the structurally related111In-1abeled analogs with improved tumor targeting and fast normal tissue and blood clearance analogs are known, specifically, for somatostatin receptor targeting68Ga-DOTA-TATE compared to111In-DOTA-TATE. Xie et al., 2021; Viola et al., 2006; Banerjee et al., 2010.

[0577] Use of the 6-fluoronicotinamide moiety has demonstrated a favorable balance of high radiochemical yield and molar activity, which can enhance the reliability of the imaging agent in10944429.601_P18646-02clinical settings. The streamlined, one-pot automated synthesis method employed here potentially reduces the production time and costs, making it more suitable for large-scale clinical applications with high molar specificity, which is comparable to the AlF-based strategies. Notably, our radiosynthesis module is routinely used for the clinical translation of developed PET radiotracers. Horti et al., 2019.

[0578] We recognized that kit-based radiolabeling is possible for A1-18F chemistry, however, none have received FDA approval. Giesel et al., 2021; Wang et al., 2021; Xie et al., 2021; Liu et al., 2019. In contrast, the 6-fluoronicotinamide prosthetic group has been successfully incorporated in PSMA-targeted18F-DCFPyL, positioning it well for clinical application of18F-FPyQCP. We and others have tried to develop an FAP-binding radiotracer without a chelated metal, including18F-1, however, most are associated with lower tumor uptake than18F-FPyQCP.

[0579] Our in-silico analysis provided valuable insight into the binding interactions of the 2-cyanopyrrolidine aminoquinolines with FAP. The docking scores suggested that the binding affinity of 1 was comparable to that of existing agents such as FAPI-04, but with some differences. The orientation of the cyanopyrrolidine moiety of18F-FPyQCP is similar to that of the OncoFAP-based compounds. Galbiati et al., 2024. The 6-fluoronicotinamide moiety provides a hydrophobic interaction involving Gln539 and Val705, thereby generating an effective fit in the large catalytic FAP pocket. That additional interaction may provide further stability of ligand bound to FAP. That interaction may be leveraged through additional modifications for future studies as needed for radiotheranostic development.

[0580] Our detailed in vivo characterization studies revealed that18F-FPyQCP might improve tumor uptake and tumor-to-background ratios compared to the most clinically translated18F-labeled agents. The biodistribution of18F-FPyQCP is similar or superior to that of previously reported FAPI compounds, with low uptake in all tissues except the kidney and bladder, consistent with predominant renal excretion. Furthermore, PET imaging data revealed that hepatobiliary clearance of18F-FPyQCP is significantly lower compared to18F-AlF-FAPI-74 in mice and nonhuman primates. Overall, the average effective whole-body dose of18F-FPyQCP PET was 1.06E-02 mSv / MBq, which is slightly lower than that of human18F-FDG PET and lower that of68Ga-F API-46 and A118F-NOTA-FAPI. Witek et al., 2024.

[0581] The comparison across different FAP expression levels in the tested tumors (U87. HT1080-FAP, and Capan-2) highlights the versatility and potential for detecting varying degrees of FAP11044429.601_P18646-02expression. That approach ensures that our agents perform strongly in diverse tumor environments, which is essential for their successful application. While human Capan-2-cells are known to be FAP-negative, this tumor model induces strong FAP-positivity in mouse CAFs and has proved a clinically relevant model to address low and heterogeneous stromal FAP expression, and has been used by other groups in similar study. Watabe et al., 2020.

[0582] The non-human primate PET imaging and dosimetry studies further reinforce the clinical potential of18F-FPyQCP, including in relation to18F-AlF-FAPI-74. The PET imaging data from a healthy male baboon shows that18F-FPyQCP exhibits lower non-specific tissue uptake, particularly in organs such as the liver, gallbladder, and intestine, which are typically associated with hepatobiliary clearance pathways. That observation highlights potentially improved specificity and reduced off-target binding of18F-FPyQCP.

[0583] Our study comes with some limitations. Although18F-FPyQCP has been purified by HPLC to remove excess precursors to ensure high molar activity, similar purification was not performed for18F-AlF-FAPI-74. A kit-based radiolabeling method was developed for18F-AlF-FAPI-74 following a reported method which is adopted for current clinical trials. Giesel et al., 2021.

[0584] Accordingly, specific molar activity for18F-AlF-FAPI-74, 18.4-25.8 GBq / mmol (500-700 Ci / mmol) is nearly two times lower than18F-FPyQCP, 92.5 GBq / mmol (approximately 2,500 mCi / mmol), which likely has some effect on tumor uptake and biodistribution of the compounds. Among the biological variables, all initial studies were conducted in male NOD / SCID mice because of the institutional availability of this mouse strain. We used nude mice, however, for head-to-head studies of18F-FPyQCP and18F-AlF-FAPI-74. Considering that most FAP-based imaging agents were evaluated in nude mice, the data generated here for18F-FPyQCP, will allow us to compare the data with relevant reported18F-labeled agents.

[0585] In summary, we have developed an18F-labeled FAP-targeted imaging agent with facile clinical translation in mind. The agent,18F-FPyQCP, could be prepared in high yield and molar activity using the time-tested 6-18F-fluoronicotinamide prosthetic group. We employed a DOTA chelator, with the presence or absence of a natural central ion for pharmacokinetic manipulation rather than for introduction of the detected radionuclide. Such a strategy ultimately provided18F-FPyQCP, which demonstrated FAP-targeted selectivity and imaging characteristics comparable to or exceeding those of18F-AlF-FAPI-74 in several realistic murine models and in a non-humanIll44429.601_P18646-02primate PET study. Streamlined preparation of18F-FPyQCP and its clinical translation are underway.

[0586] Materials and Methods

[0587] Study design

[0588] A goal of this Example was to assess18F-FPyQCP as an18F-based PET radiotracer for detecting FAP expression. We designed and synthesized three compounds with an18F-6-fluoroniconitamide moiety. We tested the FAP binding selectivity of18F-FPyQCP and related compounds in vitro and in vivo. We then demonstrated the PET imaging of18F-FPyQCP in FAP-expressing human tumor xenografts in mice. In head-to-head fashion, we also evaluated the biodistribution and PET imaging performance of18F-FPyQCP and the most heavily used investigational FAP-targeted radiotracer,18F-AlF-FAPI-74, in tumor xenografts with variable FAP expression in mice and in a healthy baboon.

[0589] Chemicals and reagents

[0590] All solvents and non-radioactive reagents were obtained in reagent grade and were used without further purification. N, N, N-Trimethyl-5-((2,3,5,6-tetrafluorophenoxy) carbonyl) pyridine-2-aminium trifluoromethane sulfonate was prepared following a reported method.Olberg et al., 2010.

[0591] Radiochemistry

[0592] Radiosyntheses of111In-1 and68Ga-1 (FIG. 8A) were carried out similarly to the previously reported procedure, Banerjee et al., 2019a; Banerjee et al., 2019b, and are included in the Supplementary Materials.18F-Fluoride was produced using a PETtrace biomedical cyclotron (General Electric, Uppsala, Sweden). The radiosyntheses of18F-1,18F-natIn-1, and18F-FPyQCP were performed using a radiochemistry synthesis box (Microlab module, General Electric). Identity of radiolabeled compounds was confirmed by co-injection with the corresponding nonradioactive reference material onto an analytical HPLC system. The final radiolabeled product was formulated as a sterile solution in saline with 8% EtOH. Details regarding the instruments and HPLC characterization of the radiotracers are provided herein below.

[0593] Synthesis of18F-l

[0594] A solution of [18F]fluoride, Kryptofix 222® (8-12 mg), and potassium oxalate (2 mg) in 1 mL of 50% aqueous acetonitrile was added to the reaction vessel of a GE MicroLab box. The mixture was heated at 120-140°C under a stream of nitrogen, while water was evaporated11244429.601_P18646-02azeotropically under vacuum after the additions of CH3CN (2x2 mL). The reactor was then cooled to 30°C. Next, 1.0 mg (0.87 pmol) of precursor 3 in anhydrous DMSO (750 pL) was added to the reactor, and the resultant solution was heated at 90°C for 10 min. The reaction mixture was then cooled to 30°C-40°C and injected onto the preparative HPLC column (cat# 186003694, Atlantis Prep T3, Waters Corporation, Milford, MA), 5 pm, 10 mmx250 mm, Mobile phase: 150:850:1 I CH3CN:water: TFA, flow rate = 8 mL / min) to collect the product (tR approcimately 19 min) in 60 mL water. The solution was then transferred to an Oasis Plus Short HLB Sep-Pak (Waters Corporation, Milford, MA). The Sep-Pak was rinsed with 10 mL saline and the product was eluted with 1 mL EtOH into a dose vial containing 4 mL saline. A second rinse of the Sep-Pak was done with saline (10 mL) into the same dose vial. The radiosynthesis time was 70-80 min. The final product was then analyzed by analytical HPLC (column Atlantis T3, 4.6x150 mm; mobile phase: 150:850:1 / CH3CN:water:TFA, flow rate = 3 mL / min; tR = 6 min) using a UV detector at 254 nm to determine the radiochemical purity and specific radioactivity at the time synthesis ended.

[0595] Synthesis of18F-FPyQCP

[0596] Preparation of crude18F-1 without HPLC purification was done as described above. The crude reaction mixture containing18F-1 was cooled to 60°C, and a solution of 3.5 mL 0.2 M sodium acetate buffer (pH=5) and 0.04 mL of 0.1 M gallium nitrate (in 0.1M HC1) was added and heated at 70-75°C for 20 min. The reaction mixture was then cooled to 30°C-40°C and injected onto the preparative HPLC column (Atlantis Prep T3, Waters Corporation, Milford, MA), 5 pm, 10x250mm, catalog # 186003694, Mobile phase: 150:850:1 / CH3CN:water: TFA, flow rate = 8 ml / min) to collect the product (tR approximately 23 min) in 60 mL water. The solution was then transferred to an Oasis Plus Short HLB Sep-Pak (Waters Corporation, Milford, MA). The Sep-Pak was rinsed with 10 mL saline and the product was eluted with 1 mL EtOH into a dose vial containing 4 mL saline. A second rinse of the Sep-Pak was done with saline (10 mL) into the same dose vial. The overall18F-FPyQCP radiosynthesis time was 100-110 min. The final product18F-FPyQCP was then analyzed by analytical HPLC (column Atlantis T3, 4.6x150 mm; mobile phase: 150:850:1 / CH3CN:water: TFA, flow rate = 3 ml / min; tR = 7.2 min) using a UV detector at 254 nm and radiation detector to determine the radiochemical purity and specific radioactivity at the time synthesis ended.

[0597] Synthesis of18F-111In-111344429.601_P18646-02

[0598] Radiosynthesis of18F-111In-1 was done similarly to18F-FPyQCP, however, instead of Ga(NO3)3a solution of In(NO3)3was used. The HPLC retention time of18F-3: preparative tR – 11.5 min; analytical tR – 4 min.

[0599] Synthesis of18F-AIF-F API-74

[0600] Synthesis of18F-AlF-FAPI-74 was executed by modification of a published method. Giesel et al., 2021. Briefly,18F-Fluoride was delivered to the hot cell in approximately 2 mL of enriched18O-H2O and was used without further purification. The automated synthesis platform was an inhouse radiochemistry box explicitly designed for radiolabeling with18F-A1F. Aqueous18F-fluoride (3.7 GBq-7.4 GBq or 100-200 mCi) was trapped on a QMA cartridge (activated using 300 mL H2O) and subsequently eluted using potassium acetate (150 mL of 10 mg / mL in H2O) in a capped V-vial, which contained AICI3 (45 ml of 0.3 mg / mL), MeCN (150 mL), ascorbic acid (50 mL of 0.5M aqueous) and FAPL74 precursor (200 mL at a concentration of 1 mg / mL in H2O). FAPL74 was prepared in-house following the reported method. Giesel et al., 2021. The reaction mixture was heated to 110°C for 12 min. The reaction was then cooled to 50 °C and diluted with ascorbic acid (6 mL of 0.5M aqueous solution) before being trapped on a Cis light SPE (activated with 2 mL EtOH, 10 mL 0.5M ascorbic acid). The product was eluted using EtOH (5 x 100 mL fractions), giving a radiochemical yield of 69 ± 5 % (n = 8, non-decay corrected). Confirmation of the product was obtained by co-elution with standard material on analytical radio-HPLC. The total synthesis time was 40-45 min. Overall radiochemical yield ranged -50-70% and specific activity was approximately 26-33 GBq / mmol (700-900 Ci / mmol).

[0601] Cell lines and culture conditions

[0602] U87 glioblastoma and Capan-2 pancreatic ductal carcinoma (PDAC) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The FAP-expressing HT-1080 cell line (FAP+ HT1080) was obtained from Dr. Zaver Bhujwalla's laboratory (Johns Hopkins). U87 and Capan-2 cells were maintained in minimum essential medium (MEM) (Coming Cellgro, Manassas, VA), containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) and 1% penicillin-streptomycin (Coming Cellgro), supplemented with sodium bicarbonate (Corning Cellgro), sodium pyruvate (Gibco, Gaithersburg, MD), and MEM non-essential amino acids (Gibco). The HT1080-FAP cell line was engineered by transducing HT1080 cells with lentivirus encoding the gene for human FAP (Accession No. NM_004460.3) that was subcloned into the lentiviral vector pMA3211. Cells were maintained in DMEM medium11444429.601_P18646-02supplemented with 10% FBS and puromycin (2 pg / mL) in a humidified atmosphere with 5% CO2 in air at 37 °C and were tested routinely for mycoplasma contamination.

[0603] In vitro studies

[0604] FAP inhibition assay

[0605] FAP, prolyl endopeptidase (PREP), and dipeptidyl dipeptidase 4 (DPPIV) inhibition assays:

[0606] The assay was conducted following our previously reported method. Boinapally et al., 2022. FAPI-04, FAPI-46 and FAPI-74 were used as positive controls. Z-Gly-Pro-AMC was used as a substrate for FAP and PREP. H-Gly-Pro-AMC was used as a substrate for DPPIV. The recombinant enzyme (0.4 pg / mL) was incubated with varying amounts of the test articles in the presence of the designated substrate (80 pM) for 10 min at room temperature. Fluorescence intensity was measured with 380 nm excitation and 460 nm emission using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT). IC50 and Ki values were obtained using a sigmoidal dose-response function. Cheng and Prusoff, 1973.

[0607] In silico molecular docking studies

[0608] The structures of compounds were built with standard bond lengths and angles using ChemDraw, and then energy was minimized with Chem3D using the integrated MM2 energy minimization script. The X-ray crystal structure of human FAP-a (PDB ID: 1Z68; resolution = 2.6 A) was retrieved from PDB. Aertgeerts et al., 2005.

[0609] Following the standard protocol, the protein and ligand PDBQT files were prepared using AutoDock Tools 1.5.6 (ADT). The following docking parameters were employed: grid box (22.5 x 22.5 x 22.5 A3) at the coordinates (x = 36.823, y = -2.170, z = 57.183): with default settings: exhaustiveness = 64, energy range = 3 kcal / mol and number of modes = 20. Docking was performed using AutoDock Vina 1.1.2 (Scripps Research Institute). Eberhardt et al., 2021. The best-scored docking pose with the lowest binding energy was selected for analysis, and figures were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC). LigPLOT+ was used to depict the 2D interactions of the ligand and the protein.

[0610] Mo use xenograft models

[0611] Animal studies were performed according to protocols approved by the Johns Hopkins University Animal Use and Care Committee. Male NOD / SCID mice 6-8 weeks old were11544429.601_P18646-02purchased from the Johns Hopkins Research Animal Resources (Baltimore, MD). NOD / SCID mice were subcutaneously inoculated in the upper right flank with 3 million of either U87, or Capan-2 or HT-1080-FAP in HBSS. Mice were monitored for tumor size and used for imaging or biodistribution when the tumors reached 100-150 mm3.

[0612] Tissue biodistribution

[0613] Biodistribution studies were conducted following our previously reported method. Banerjee et al., 2019a; Banerjee et al., 2021. Tumor-bearing mice were administered with 0.74 ± 0.07 MBq (20 ± 2 pCi) in 150 pL saline via tail-vein injection. Mice were euthanized at the indicated time after the injection, and blood, tumor, and selected organs (heart, lungs, liver, stomach, pancreas, spleen, fat, kidney, small intestine, salivary gland, lacrimal gland, urinary bladder, bone, muscle) were harvested, weighed, and assayed for radioactivity using an automated y-counter. The percentage of injected dose per gram of tissue (% ID / g) was calculated by comparison with samples of a standard dilution of the initial dose.

[0614] Small-animal PET / MR imaging

[0615] A simultaneous PET / MR scanner with a 3 ring Bruker Si 198 PET insert (7T preclinical PET / MRI, Bruker BioSpec, 70 / 30 USR, Germany) was used. Tumor-bearing mice were injected with 7.4 ± 0.4 MBq (200 ± 10 pCi) of the indicated radiotracer intravenously, with anesthesia induced under 3% isoflurane and maintained under 1.5% isoflurane (v / v) for imaging studies. Static and dynamic images (two-bed positions, 10 min per position) were acquired at indicated time points after injection.

[0616] Flow cytometry, and immunohistochemistry (IHC)

[0617] Flow cytometry, and IHC were performed following our previous report. Banerjee et al., 2019a; Banerjee et al.. 2021.

[0618] Non-human primate PET imaging study using18F-FPyQCP and dosimetry

[0619] Whole-body PET / CT was performed on a Siemens Biograph mCT system (Siemens Healthineers. Knoxville, TN, USA). Sequential whole-body dynamic PET images were acquired at eight time points over 1.5 hours, starting immediately after bolus intravenous injection of18F-FPyQCP or18F- A1F-FAPI-74 in an adult baboon (Papio anubis) one wk apart. The injected doses of radioactivity were 212.74 MBq (5.74 mCi), molar activity 1,096-1,184 GBq / pmol (29.6-32.0 Ci / pmol) for [18F]FPyQCP and 246.42 MBq (6.6 mCi) with molar activity 1100 GBq / pmol, for18F-AlF-FAPI-74, respectively. Adult male baboons weighted 24.5-26 kg were used for dosimetry.11644429.601_P18646-02PMOD (v3.7, PMOD Technologies Ltd, Zurich, Switzerland) was used for image processing. The Rapid 3D-RD software (Rapid Inc, Baltimore, MD, USA) was used to calculate organ absorbed doses and the effective dose.

[0620] Statistics

[0621] To assess the changes between two radiotracer uptake we performed statistical analyses in two ways: (i) Student’s t test was used to compare quantitative data between two independent samples; and (ii) for comparisons involving more than two categories, two-way ANOVA, followed by a Sidak’s multiple comparison test was performed (GraphPad Prism 10.3 San Diego, CA). P-values are indicated by asterisks as *P < 0.05, **P < 0.01, ***P < 0.0001. Quantitative values were expressed as means ± SD. IC50 values were determined using nonlinear regression curve fit, variable slope, and least- squares fit.EXAMPLE 2

[0622] Experimental

[0623] Radiosynthesis of [18F]-natGa-1 (18F-FPyQCP)

[0624] Preparation of crude18F-1 without HPLC purification was done as described above. The crude reaction mixture containing18F-1 was cooled to 60 °C, and a solution of 3.5 mL 0.2 M sodium acetate buffer (pH = 5) and 0.04 mL of 0.1 M gallium nitrate (in 0.1M HC1) was added and heated at 70°C - 75 °C for 20 min. The reaction mixture was then cooled to 30 °C - 40 °C and injected onto the preparative HPLC column (Atlantis Prep T3, Waters Corporation, Milford. MA), 5 pm, 10 mm x 250 mm, part # 186003694, Mobile phase: 150:850:1 / CHsCNtwaterTFA, flow rate = 8 mL / min) to collect the product peak (tR approximately 23 min) in 60 mL water. The solution was then transferred to an Oasis Plus Short HLB Sep-Pak (Waters Corporation, Milford, MA). The Sep-Pak was rinsed with 10 mL saline and the product was eluted with 1 mL EtOH into a dose vial containing 4 mL saline. A second rinse of the Sep-Pak was done with saline (10 mL) into the same dose vial. The overall18F-FPyQCP radiosynthesis time was 100 min - 110 min. The final product18F-FPyQCP was then analyzed by analytical HPLC (column Atlantis T3, 4.6 mm x 150 mm; mobile phase: 150:850:1 / CH3CN:water:TFA, flow rate = 3 mL / min; tR = 7.2 min) using a UV detector at 254 nm and radiation detector to determine the radiochemical purity and specific radioactivity at the time synthesis ended. The radiochemical yield of18F-FPyQCP was approximately 25%. radiochemical purity greater than 95% and molar (specific) radioactivity was 2000 - 5000 Ci / mmol.11744429.601_P18646-02

[0625] Radiosyntheses of [18F]-natIn-l and [18F]-natSc-l were performed similarly to that of [18F]-natGa-l (18F-FPyQCP) using solutions ofnatInC13 ornatScCl3(or nitrates) instead ofnatGa(NC>3)3.

[0626] Radiosynthesis of [68Ga]-1 (68Ga-FPyQCP)

[0627] Eckert and Ziegler68Ge / 68Ga-generator was used as a68Ga source.68Ga was eluted using 0.5 N HC1 solution. TraceSELECT grade water and sodium acetate were used for radiolabeling. Radiolabeling was performed with a minor modification of our reported method. Ray Banerjee et al., 2016. To a reaction vial (5 m ) containing 600 pL of68Ga(III) (approximately 226 MBq) in 0.1 HC1 was neutralized to pH 4 using 15 pL of 5 M ammonium acetate and 10 pL of compound 1 (2 mM solution). The reaction vial was heated at 95 °C for 10 min. The solution was then cooled, diluted with 200 pL water, and purified by HPLC to provide 172 MBq of68Ga-FPyQCP (Table 16). The flow rate was 1 mL / min, with water (0.1% trifluoroacetic acid) (A) and CH3CN (0.1% trifluoroacetic acid) (B) as the eluting solvents, respectively. An isocratic solution of 75% / 25% (A / B) was used to separate the excess ligand from the radiolabeled compound and to ensure the highest purity. HPLC retention time ( / R) = 16.4-18.8 min for the radiolabeled productand = 13.2-14.4 min for the free ligand. The HPLC eluted solution was diluted with 20 mL water and trapped on a Cis SPE (activated with 2 ml CH3CN and 2 mL water). The column was eluted with 2 x 2 mL water followed by 1 mL ethanol. The ethanol eluate was reduced under nitrogen on a hot plate at 65 °C to dryness. The solid residue was diluted with desired volume of 0.9% sterile saline (approximately 500 pL) and 50 pL sodium carbonate (1 M). This formulated solution was used in all biological studies with indicated amount of radioactivity.Table 16. HPLC Methods for68Ga-l and111In-1Metal HPLC Retention %A (0.1% TFA in H2O) %B (0.1% TFA in time MeCN)111In-1 13.6 min 85 1568Ga-l 13.4 min 83 17

[0628] FAP potency and selectivity

[0629] The potency and selectivity was determined by performing competitive inhibition assay of the test compounds peptidase activities of FAP (R& D Systems, Catalog # 3715SE), DPPIV ) R& D11844429.601_P18646-02Systems, Catalog # 9168-SE), and PREP (R& D Systems, Catalog # 4308-SE). The test compound was diluted in Assay buffer (test concentrations: 80 pM, 8 pM, 800 nM, 80 nM, 8 nM, 0.8 nM, 0.08 nM, and 0.008 nM). The rhFAP was diluted in Assay Buffer (1.6 pg / mL). The substrate was diluted in Assay Buffer (200 pM). 25 pL of the test compounds was added into a plate. 25 pL of the enzyme was added into a plate and the reaction was started by adding 50 pL of substrate. The plate was incubated for 10 minutes at room temperature. Fluorescence intensity was measured with 380 nm excitation and 460 nm emission. All assays were performed in triplicates. IC50 values were determined from semi-log plots of inhibitor concentration of FPyQCP versus residual enzyme activity of FAP (sigmoidal dose-response), representing the concentration at which enzyme activity was inhibited by 50%. All assays were performed in triplicate, and Ki values were calculated using the Cheng-Prusoff equation. Cheng and Prusoff, 1973.

[0630] GraphPad Prism 10 software was used for the analysis.

[0631] Assay Buffers: FAP (50 mM Tris, IM NaCl, 1 mg / mE BSA, pH 7.5), DPPIV (25 mM Tris, pH 8.0), PREP (25 mM Tris, 250mM NaCl. 2.5 mM Dithiothreitol, pH 7.5)

[0632] Substrate: FAP and PREP (Z-Gly-Pro-AMC (Bachem, Catalog # 11145), DPPIV (H-Gly-Pro-AMC (Bachem, Catalog # 11225).

[0633] Summary’

[0634] In summary, we have developed an18F-labeled FAP-targeted imaging agent with facile clinical translation in mind. The agent, [18F]-natGa-l (18F-FPyQCP), could be prepared in high yield and molar activity using a 6-18F-fluoronicotinamide prosthetic group. We employed a DOTA chelator, with the presence or absence of a natural metal cation for pharmacokinetic manipulation rather than for introduction of the detected radionuclide. Such a strategy ultimately provided18F-FPyQCP, which demonstrated FAP-targeted selectivity and imaging characteristics comparable to or exceeding those of18F-AlF-FAPI-74 in several realistic murine models and in a non-human primate PET study. In addition, [68Ga]l (68Ga-FPyQCP) also was synthesized and manifested similar in vivo properties in mice bearing U87 xenografts to those of18F-FPyQCP.EXAMPLE 3

[0635] 18F-FPyQCP, a PET imaging agent for detecting fibroblast activation protein

[0636] Synthesis and characterization

[0637] (9ff-FIuoren-9-yI) methyI((5)-3-amino-4-((3-((4-((2-((5)-2-cyanopyrrolidin-l-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-4-oxobutyl)carbamate (4): To a stirred11944429.601_P18646-02solution of FAP-NH2 (100 mg, 0.262 mmol, 1.0 eq), Boc-Dab(Fmoc)~OH (127 mg, 0.288 mmol, 1.1 eq) and HATU (119.7 nig, 0.315 mmol, 1.2 eq) in DMF (1 mL) was added DIPEA (160 pL, 0.918 mmol, 3.5 eq) at room temperature. The reaction mixture was stirred for 2 h and concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a MeCN / water / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, desired compound (185 mg, 88%) was obtained as a pale-yellow solid. To the stirred solution of above compound was added 2 mL of TFA / CH2CI2 (1:1) at room temperature and mixture was stirred for 1 h. Reaction mixture was concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a MeCN / HzO / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, 4 (145 mg, 90%) was obtained as a pale- yellow solid.fH-NMR (500 MHz, DMSO-tfc): d 9.05 (t, J - 6.0 Hz, 1H), 8.84 (d, 7- 4.0 Hz, 1H). 8.57- 8.50 (m, 1H), 8.20-8.08 (m, 3H), 8.00 (d, J = 9.5 Hz, 1H), 7.88 (d, J - 7.5 Hz, 2H), 7.66 (d, J 7.0 Hz. 2H), 7.55 (d, J = 4.0 Hz, 1H), 7.48 (d,,7- 8.5 Hz. 1H0. 7.41 (t, 7 = 7.5 Hz, 2H), 7.35-7.28 (m, 2H), 4.82-4.77 (m, 1H). 4.34 (d, J = 6.0 Hz. 2H), 4.20 (s, 4H). 3.75-3.64 (m, 2H), 3.53 (q, J = 8.0 Hz, 1H), 3.41-3.29 (m, 2H), 3.09-3.00 (m, 2H), 2.30-1.71 (m, 10H); ESLMS: m / z calculated for C39H41N7O6 [M + H]+703.80; found 704.7.

[0638] Tri -tert- butyl 2,2',2”-(10-(2-(((S)-4-amino-l-((3-((4-((2-((5)-2-cyanopyrrolidin-l-yI)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-1-oxobutan-2-yl)amino)-2- oxoethyl)-l,4,7,10-tetraazacyclododecane-l,4>7-triyl)triacetate (2): To a stirred solution of 4 (103 mg, 0.146 mmol, 1.0 eq), DOTA-tris (t-Bu ester) (92.3 mg, 0.161 mmol, 1.1 eq) and HATU (61.28 mg, 0.161 mmol, 1.1 eq) in DMF (1 mL) was added DIPEA (90 p L, 0.512 mmol, 3.5 eq) at room temperature. The reaction mixture was stirred for 2 h and concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a MeCN / water / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, desired compound (143.6 mg, 78%) was obtained as a pale-yellow solid. To the stirred solution of above compound was added 20% piperidine in DMF (1 mL) at room temperature and mixture was stirred for 1 h. Reaction mixture was concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a MeCN / H2O / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, 2 (101.5 mg, 86%) was obtained as a pale-yellow solid. 1H-NMR (500 MHz, DMSO-d6): δ 9.06 (t, J = 6.0 Hz, 1H), 8.83 (d. J = 4.0 Hz, 1 H). 8.31-8.26 (m, 1 H), 8.00 (d, J = 9.0 Hz, 1H), 7.91-7.82 (m, 3H), 7.55 (d, J = 4.0 Hz, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.40-12044429.601_P18646-027.32 (m, 1H), 4.83-4.77 (m, 1 H), 4.23-4.12 (m, 8H), 3.77-3.69 (m, 1 H), 3.56-3.04 (m, 16H), 3.00- 2.75 (m, 8H). 2.30-1.75 (m, 10H), 1.47 (s. 9H), 1.40 (s, 9H), 1.37 (s, 9H); ESI-MS: m / z calculated for C52H81N11O11 [M + H]+1036.29; found 1037.7.

[0639] 2,2',2"-(10-(2-(((S)-l-((3-((4-((2-((S)-2-cyanopyrrolidin-l-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-4-(6-fluoronicotinamido)-l-oxobutan-2-yl)amino)-2-oxoethyl)-l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid (1): To a stirred solution of 2 (50.1 mg. 48.35 pmol. 1.0 eq), 6-Fluoronicotinic acid (8.2 mg, 58.0 urnol, 1.2 eq) and HATH (22 mg. 58.0 pmol, 1.2 eq) in DMF (1 tnL) was added DIPEA (29.4 pL, 169.25 pmol, 3.5 eq) at room temperature. The reaction mixture was stirred for 2 h and concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Star, UK). The product was purified with a MeCN / water / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, required compound (47.6 mg, 85%) was obtained as a pale-yellow solid. To the stirred solution of above compound was added 1 mL cocktail of TFA: H2O: TIPS (95: 2.5: 2.5) at room temperature and mixture was stirred for 4 li. Reaction mixture was concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a MeCN / H2O / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, 1 (31 mg, 76%) was obtained as a pale-yellow solid.JH-NMR (500 MHz, DMSO- tf): 9.04 (t, J = 5.5 Hz, 1H), 8.82 (cl, J = 4.5 Hz, 2H), 8.70-8.60 (m, 2H), 8.33 (t, J - 8.5 Hz, I M). 8.27-8.20 (m, 1H), 7.98 (d, J = 9.5 Hz, 1H), 7.85 (s, 1H), 7.53 (d, 7 = 4.5 Hz, 1H), 7.46 (d, J = 7.0 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 4.85-4.77 (m, 1H), 4.37-4.30 (m, 1H), 4.23-4.14 (m. 4H). 4.10-3.97 (m, 3H). 3.76-3.69 (ra, 1H), 3.58-3.50 (m, 3H), 3.45-3.21 (m, 13H), 3.08 (s, 8H), 2.27-2.00 (m, 6H), 1.94 (t,. / = 5.5 Hz, 2H). 1.87-1.69 (rn. 2H): HRMS (ESI) m / z: [M + H]+ calcd for C46H60FN12O12. 991.4431: found, 991.4432.

[0640] natGa-l: To the solution of gallium (III) nitrate hydrate (2.58 mg, 10.09 pmoL 5.0 eq) in NHiOAc buffer (600. L) at pH approximately 4 was added 1 (2.0 mg, 2.01 pmol, 1.0 eq) at room temperature. The reaction mixture was stirred for 15 min at 90 °C and purified by preparative RP- HPLC chromatography using 0.1% TFA in H2O and 0.1% TFA in acetonitrile as eluents followed by lyophilization affordednatGa-l(2.0 mg, quantitative yield) as a white solid. [RP-HPLC purification was achieved using Agilent System, A.254 nrn, 250 mm. x 10 mm Phenornenex Luna C18 column, solvent gradient: 98% H2O (0.1% TFA) and 2 % ACN (0.1% TFA). reaching 35%; of ACN in 0 to 25 min, 90% of ACN in 25 to 28 min at a flow rate of 10 mL / min, product eluted12144429.601_P18646-02at 13.8 min]; ESI-MS: m / z calculated for C^HseFGaNnOn [M + H]+1057.3453; found 1058.3457. 552+56+18+168+192.

[0641] (9H-fluoren-9-yl)methyl ((R)-3-amino-4-((3-((4-((2-((S)-2-cyanopyrrolidin-l-yi)-2- oxoethyl)carbamoyI)quinolin-6-yl)oxy)propyl)amino)-4-oxobutyI)carbamate (5): To a stirring solution of FAP-NH2 (50 mg, 0.13 mmol, 1.0 eq.), Boc-D--Dab(Fmoc)-OH (64 mg, 0.14 mmol, 1.1 eq.) and HATU (60 mg, 1.2 eq.) in DMF (0.5 mL), DIPEA (80 pL, 0.46 mmol. 3.5 eq.) was added and the mixture allowed to stir 2 h at room temperature. After removal of solvent under vacuum, 2 mL of TFATTECb (1:1) was added and stirred for another 30 min. The mixture was then concentrated and loaded onto a 12 g C18 cartridge (Biotage Sfar). The product was purified with a MeCN / HjO / TFA gradient (5 / 95 / 0.1 to 50 / 50 / 0.1). After lyophilization, 73.6 mg of 5 was obtained as a white solid in 92% yield.

[0642] tri-tert-butyl 2,2',2"-(10-(2-(((R)-4-amino-l-((3-((4-((2-((S)-2-cyanopyrroHdin-l-yI)-2~oxoethyl)carbaaioyi)quinolin-6-yl)oxy)propyI)amino)-l-oxobutan-2«yl)amino)-2~ oxoefliyl)-l,4,7, W"tetraazacyclododecane-l,4,7-triyl)triacetate (6): To a stirred solution of 5 (24 mg, 0.03 mmol, 1.0 eq.) and DOTA-tris (;Bu ester) (31 mg, 0.04 mmol. 1.1 eq.) in DMF (500 pL) was added DIPEA (30 pL, 0.17 mmol, 5.0 eq.) at room temperature. The reaction mixture was stirred for 2 h and purified using MeCN / HiO on Cis Sep-Pak column to provide required product as a white solid (33 mg, 78%). To the above product (27 mg, 0.026 mmol) was added 2 mL of 20% piperidine at room temperature, this mixture was stirred for 1 h and then concentrated and purified using MeCN / HiO on Cis Sep-Pak column to provide compound 6 (22.8 mg. 82%) as a white solid.

[0643] 2,2',2"-(10-(2-(((R)-l-((3-((4-((2-((S)-2-cyanopyrrolidin-l-yl)-2-oxoethyI)carbamoyl)quinolin-6-yl)oxy)propyl)amino)-4-(6-fluoronicotinamido)-l-oxobutan-2-yI)amino)-2-oxoethyI)-l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid (D-l): To a stirring solution of 6 (15 mg, 14.5 mmol, 1.0 eq.), 6- fluoronicotinic acid (2.7 mg, 18.8 mmol, 1.3 eq.) and HATU (7.2 mg, 18.8 mmol, 1.3 eq.) in DMF (300 uL), DIPEA (8.8 pL, 50.7 mmol, 3.5 eq.) was added. The mixture was allowed to stir for 2 h at room temperature. After removal of solvent under vacuum, 2 mL of TFA: CH2C12 (1:1) was added and stirred for another 30 min. The mixture was then concentrated and loaded onto a 12 g C18 cartridge (Biotage Sfar). The product was purified with MeCN / FUO / TFA gradient (5 / 95 / 0.1 to 50 / 50 / 0.1). After lyophilization, 9.6 mg of D-l was obtained as a white solid in 80% yield. 'H NMR (500 MHz,12244429.601_P18646-02DMSO) 89.05 (t, J = 6.0 Hz, 1 H), 8.82 (d, J = 4.3 Hz, 2H), 8.66 (d, J = 10.4 Hz, 2H), 8.38 - 8.30 (m, 1H), 8.25 (s, 1H), 7.99 (d, J= 9.2 Hz, 1H), 7.86 (s, 1H), 7.54 (t, J= 5.6 Hz, 1H), 7.47 (dd, J = 9.2, 2.4 Hz, 1H), 7.29 (dd, J = 8.5, 2.3 Hz, 1H), 4.81 (dd, J = 7.7, 3.4 Hz, 1H), 4.37 - 4.30 (m, 1H), 4.19 (dt, J= 12.1, 5.8 Hz, 4H), 4.03 (d, J= 32.6 Hz, 5H), 3.74 (dd, J = 11.4, 5.6 Hz, 9H), 3.54 (dd, J= 16.2, 8.5 Hz, 5H). 3.37 (dd, J = 14.4, 7.3 Hz, 10H), 3.26 (dd, J = 12.7, 6.2 Hz, 3H), 3.08 (s. 4H), 2.24 - 2.13 (m, 1H), 2.08 (d, J = 4.8 Hz, 2H), 2.02 (d, J = 7.8 Hz. 2H), 1.99 - 1.90 (m, 2H), 1.81 (d, J= 8.5 Hz, 1H); MS (ESP ): mfr. [M + H+] 991.4493; ealed for C46H59FN12O12: 990.4400.H2NAYN'

[0644] Scheme 3. Synthesis of I andnatGa-D-l.

[0645] 5-(((S)-4-((3-((4-((2-((S)-2-Cyanopyrrolidin-l-yl)-2-oxoethyl) carbamoyl) quinolin-6-yl) oxy) propyl) amino)-4-oxo-3-(2-(4,7,10-tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl) acetamido) butyl) carbamoyl)-N, N, N-trimethylpyridin-2-aminium (3, precursor): To a stirred solution of 2 (45 mg, 43.47 pmol, 1.0 eq) and A,7V, Af- trimethyl-5-((2,3,5,6-tetrafluorophenoxy) carbonyl)pyridin-2-aminium trifluoromethanesulfonate (22.8 mg, 47.82 pmol, 1.1 eq) in DMF (500 jiL) was added DIPEA (30 pL, 173.9 pmol, 4.0 eq) at room temperature. The reaction mixture was stirred for 2 h and concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a MeXIN / H O / TFA gradient (0 / 100 / 0.1 to 90 / 10 / 0.1). After lyophilization, desired compound (46 mg, 89%) was obtained as a pale-yellow solid. To the stirred solution of above compound was added 1 mL cocktail of TFA: H2O: TIPS (95: 2.5: 2.5) at room temperature and mixture was stirred for 4 h. Reaction mixture was concentrated to get the crude which was loaded onto a 30 g C18 cartridge (Biotage Sfar, UK). The product was purified with a Me. CN / H2O / TFA gradient12344429.601_P18646-02(0 / 100 / 0.1 to 90 / 10 / 0.1 ). After lyophilization, 3 (32 mg, 81 %) was obtained as a pale-yellow solid. H-\ R (500 MHz, D. MSO-A- ): d 9.04 (t, J - 6.0 Hz, 1H), 8.97 (d, J 2.0 Hz, 1H), 8.89 (t, J = 5.5 Hz, 1H), 8.81 (d. J = 4.5 Hz, 2H), 8.51 (dd, J = 2.0, 8.5 Hz, 1H). 8.24 (t,.7= 5.0 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1 H), 7.98 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 2.5 Hz, 1 H). 7.53 (d,. / - 4.5 Hz, 1H), 7.46 (dd, J - 2.0, 9.0 Hz, 1 H ), 4.81 (dd, J - 3.5, 8.0 Hz, 1H), 4.38-4.30 (m, 1H), 4.20 (d,. / - 6.5 Hz, 2H), 4.16 (t, J = 6.0 Hz. 2H), 4.09-3.93 (m, 4H), 3.76-3.69 (rn, 1H), 3.59 (s. 9H), 3.54 (q. J = 7.0 Hz, 3H), 3,44-3.23 (m, 12H), 3.08 (s, 8H), 2.27-2.14 (m, 2H), 2.12-1.89 (m, 6H), 1.86- 1.77 (m, 1H), 1.29-1.17 (m, 1H); ESI-MS: m / z calculated for C49H68Ni3Oi2+[M]+1031.16; found 1030.7.ln(NO3)3, 02 M Ammonium acetate buffer pH -50, 70 oC, 30 min, quantitative

[0646] Scheme 4. Synthesis ofnatIn-l.

[0647] natIn-l: To the Indium (III) nitrate hydrate (100 mM solution in 0.1 M HC1. 100 pL. 5.0 eq) in NH4OAC buffer (0.2 M solution, 1 mL) at pH approximately 5.0 was added solution of 1 in DMSO (2.0 mg in 50 pL DMSO, 1.0 eq) at room temperature. The reaction mixture was stirred for 45 min at 60 °C and purified by preparative RP-HPLC chromatography using 0.1% TFA in H2O and 0.1% TFA in acetonitrile as eluents followed by lyophilization affordednatIn-l (quantitative yield) as a white solid. [RP-HPLC purification was achieved using Agilent System, A, 254 nm, 250 mm. x 10 mm Phenomenex Luna C18 column, solvent gradient: 98% H2O (0.1% TFA) and 2 % ACN (0.1% TFA), reaching 30% of ACN in 0 to 20 min, 90% of ACN in 20 to 25 min at a flow rate of 10 mL / min. product eluted at 1.2 min]; HRMS (ESI) m / z: [M + H]+ calcd for C kl hrX-Oi.'.. 1103.3230; found, 1103.3236.

[0648] Radiolabeling

[0649] The high-performance liquid chromatography (HPLC) system consisted of Waters model 600 pumps, Rheodyne model 7126 injectors, an in-line Waters model 441 UV detector (254 nm) and a Bioscan FC-3200 Nal detector connected to an FC- 1000 base unit. All HPLC chromatograms were recorded and analyzed with Varian Galaxy Chromatography Data System software. A dose calibrator (Capintec 15R) was used for all radioactivity measurements.12444429.601_P18646-02

[0650] Preparative HPLC column

[0651] For the purification of the18F-labeled compounds, Atlantis T3, 10*250 mm (Waters) preparatory column and analytical column Atlantis T3, 5 me, 4.6*150 mm (Waters) were used. For68Ga andinIn-labeled compounds, HPLC purification was performed on an Agilent HPLC instrument (Agilent Technologies, Santa Clara, CA) coupled to a radiodetector (Bioscan - Flow-Count). using a Phenomenex Luna Cis HPLC column (00G-4252-E0), 250 x4.6 mm, 5p, 100A. The purified radiolabeled product was diluted with saline to the desired radioactivity concentration for imaging and biodistribution studies.

[0652] Flow Cytometry

[0653] U-87, Capan-2, HT-1080 and HT-1080-FAP cells (1 x 106cells) were harvested using cell dissociation buffer (Gibco) and transformed into a single-cell suspension. The harvested cells were washed twice with flow cytometry buffer (lx phosphate-buffered saline with 2 mM ethylenediaminetetraacetic acid and 0.5% fetal bovine serum) and were passed through a 70 pm strainer by pipetting. Next, the cells were stained with PE anti-FAP antibody (cat#342504, BioLegend) following the manufactured protocol. The cells were incubated at 4 °C for 30 minutes in the dark, followed by washing with cold PBS. The fluorescence intensities of both unstained and stained cells were analyzed using flow cytometry (BD), and quantitative data analysis was performed with FlowJo software.

[0654] Immunohistochemistry

[0655] Immunohistochemical staining was conducted at the Oncology Tissue Services Core at Johns Hopkins University following our previously reported method. Boinapally et al., 2022.

[0656] Formalin-fixed, paraffin-embedded tissue sections were subjected to immunolabeling for FAP. The procedure began with dewaxing and rehydration, followed by epitope retrieval using Ventana Ultra CC1 buffer (catalog# 6414575001, Roche Diagnostics) at 96°C for 64 minutes. The primary antibody, anti-FAPa (1:100 dilution; catalog# ab227703, Abeam), was applied at 36°C for 60 minutes. Detection of the primary antibodies was carried out using an anti-rabbit HQ detection system (catalog# 7017936001 and 7017812001, Roche Diagnostics), with signal amplification via the Discovery AMP Multimer (catalog # 6442544001, Roche Diagnostics). The staining was visualized using the Chromomap DAB IHC detection kit (catalog # 5266645001, Roche Diagnostics), followed by counterstaining with Mayer’s hematoxylin, dehydration, and mounting.12544429.601_P18646-02

[0657] P ET data analysis (murine study)

[0658] All dynamic PET analyses were performed by PMOD (V3.5, PMOD Technologies, Basel, Switzerland) using MRI (tumor) and tracer-specific templates for spatial normalization. The average FAP-PET VOI / ml was obtained from a spherical volume of interest at the tumor site as the primary PET readout (16). Within the tumor volume of interest, we performed a 50-step k-means clustering using the PMOD segmentation tool (10 iterations) to delineate individual clusters of tumor tracer uptake in each tumor.

[0659] PET data analysis (Baboon study)

[0660] The dynamic PET protocol included 8 repeated whole-body scan frames to cover each participant from vertex to mid-thigh. The time per bed position and scan starting time for each frame were 1.5 min / bed at 0.01 h p.i., 0.11 h p.i. and 0.22 h p.i. (frames 1 to 3), 3.0 min / bed at 0.33 h p.i., 0.54 h p.i. and 0.76 h p.i. (frames 4 to 6), 4.5 min / bed at 0.97 h p.i. and 1.28 h p.i. (frames 7 and 8), respectively. A low-dose CT scan was also performed for attenuation correction. The images were reconstructed using OSEM algorithm with correction for attenuation, scatter, randoms, and time-of-flight. A total of 2 iterations with 21 subsets per iteration were used.

[0661] The PMOD (v3.7, PMOD Technologies Ltd, Zurich, Switzerland) was used for image processing. The Rapid 3D-RD software (Rapid Inc, Baltimore, MD, USA) was used to calculate organ absorbed doses and the effective dose. Sixteen organs-of-interest were manually delineated on each co-registered CT image by a trained medical physicist. The region of interest (ROIs) were then applied to the corresponding PET images to obtain time-activity curves (TACs) that were generated without decay correction. The TACs from baboons were then converted to standard ICRP 89 adult male model based on body weight. Image-derived time-activity data were fitted using a bi-exponential or a single exponential model and analytic integration was used to estimate the time-integrated activity coefficient (TIAC), the total number of disintegrations in the organ divided by the administered activity.EXAMPLE 4

[0662] This Example provides the synthesis and characterization of compounds of formula (I). In some embodiments, the non-radiolabeled standards 1,nalIn-l,natSc-l andnatGa-l, the structures of which are provided immediately herein below, were synthesized:12644429.601_P18646-02

[0663] The in vitro FAP potency and selectivity of the new series were determined (Table lb). Radiotracers [18F]1, [18F]-natIn-l, [18F]-natSc-l and [18F]-natGa-l (FIG. 3a) were synthesized via the trimethylammonium precursor 3.

[0664] Scheme 5. Synthesis of radiotracers [18F]1, [18F]-natIn-l, [18F]-“atSc-l and [18F]-natGa-l via the trimethylammonium precursor 3.

[0665] The precursor 3 was prepared as shown in Scheme 2 hereinabove:12744429.601_P18646-02

[0666] The imaging properties of [18F]1, [18F]-natIn-l, [18F]-natSc-l and [18F]-natGa-l were tested in murine U87 glioblastoma model in NOD / SCID mice, data provided in Tables 7-9, and FIG. 5i, in Example 1, provided herein below. Among the studied radiotracers, the natural gallium chelate [18F]-natGa-l exhibited the highest uptake in the U87 tumor and high tumor-to-organ ratios. The U87 binding of the radiotracers was highly FAP- specific as demonstrated in the blocking experiments (blocker - unlabeled FAPI-04, 30 nmol / mouse). Noteworthy, for [18F]-natGa-l the U87 baseline / block ratio was greater than that of the established FAP radiotracer Al18F-FAPI-74 (40 and 18, respectively) (see Table 10a and Table 10b).

[0667] Compound [18F]-natGa-l (18F-FPyQCP) also was compared with the clinical FAP tracer,18F-AlF-FAPI-74, Giesel et al., 2021; Dahl et al., 2021; Wang et al., 2021; Lindner et al., 2021; Liu et al.. 2021, head-to-head in the same batch of male nude mice bearing U87 xenografts (Table 10a and Table 10b). This comparison study demonstrated that18F-FPyQCP achieved significantly higher tumor uptake alongside improved biodistribution. Specifically, biodistribution and PET imaging revealed rapid blood and normal tissue clearance for18F-FPyQCP with higher tumor-to-organ ratios in most organs compared to A118F-FAPL74. The chemical structures of radiolabeled isotopomers [18F]-natGa-l ([18F]FPyQCP) and [68Ga]1([68Ga]FPyQCP) are provided immediately herein below:

[0668] In addition to [18F]-natGa-l (18F-FPyQCP), we radiolabeled and tested in mice its Ga-68 isotopomer [68Ga]l (68Ga-FPyQCP) (Table 10a and Table 10b). The mouse studies demonstrated similar properties of the18F and68Ga isotopomers (compare Tables 5a-5b and Tables 9a-9b).12844429.601_P18646-02<[68Ga]FPyQCP)

[0669] Scheme 6. Radiosynthesis of [68Ga]l (68Ga-FPyQCP).

[0670] Summary

[0671] In summary, we have developed an18F-labeled FAP-targeted imaging agent with facile clinical translation in mind. The agent, [18F]-natGa-l (18F-FPyQCP), could be prepared in high yield and molar activity using a 6-18F-fluoronicotinamide prosthetic group. We employed a DOTA chelator, with the presence or absence of a natural metal cation for pharmacokinetic manipulation rather than for introduction of the detected radionuclide. Such a strategy ultimately provided18F-FPyQCP, which demonstrated FAP-targeted selectivity and imaging characteristics comparable to or exceeding those of18F-AlF-FAPI-74 in several realistic murine models and in a non-human primate PET study. In addition, [68Ga]l (68Ga-FPyQCP) also was synthesized and manifested similar in vivo properties in mice bearing U87 xenografts to those of18F-FPyQCP.REFERENCES

[0672] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

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[0765] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.14044429.601_P18646-02

Claims

THAT WHICH IS CLAIMED:

1. A compound of formula (I):XLbC-La-A(i);wherein:A is a targeting moiety for fibroblast activation protein alpha (FAP-a);C is a chelating group comprising a naturally occurring non-radioactive isotope of a metal;Pis a prosthetic group;X is a radioisotope of a halogen;Lais a tri-functionalized linker capable of forming a chemical bond with A, Lb, and C; Lb is a bi-functionalized linker capable of forming a chemical bond with Laand P; and stereoisomers and pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein A comprises an FAP-a targeting moiety having the following structure:(A’);wherein each y is independently an integer selected from 0, 1, and 2;Rix, R2X, and Rax', are each independently selected from H, OH, halogen, Ci-ealkyl, -O-Cn ealkyl, and -S-Ci-ealkyl;Raxis selected from H, -CN, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and 5-tetrazolyl;14144429.601_P18646-02R4X is selected from H, straight-chain or branched C1-6 alkyl, -(CH2)q4-aryl, and hydroxylsubstituted straight-chain or branched C1-6 alkyl, wherein q4 is an integer selected from 0, 1, 2, 3, 4, 5, and 6;Rs, Rex, and R?xare each independently selected from H, -OH, oxo, halogen, -Ci-ealkyl, -O-Ci-6alkyl, -S-Ci-6alkyl, -NRsxRyx, -ORi2x, -Het2and -Ar2; each of Ci-6alkyl being optionally substituted with from 1 to 3 substituents selected from -OH and halogen;Rs, Ryx, and RI2Xare each independently selected from H, -OH, halo, -Ci-ealkyl, -O-Ci-ealkyl, -S-Ci-6alkyl, and -Ar3;Riox, Riix, Ri3x and Ri4x are each independently selected from H, -OH, halogen, -Ci-ealkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl; An, Ar2and Ar3are each independently a 5- or 6-membered aromatic monocycle optionally comprising 1 or 2 heteroatoms selected from O, N and S; each of An, Ar2and Ar3being optionally and independently substituted with from 1 to 3 substituents selected from -NRioxRiix, -Ci-ealkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl;Het2is a 5- or 6-membered non-aromatic monocycle optionally comprising 1 or 2 heteroatoms selected from O, N and S; Het2being optionally substituted with from 1 to 3 substituents selected from -NRi3xRi4x, -Ci-6alkyl, -O-Ci-6alkyl, and -S-Ci-6alkyl;v is 0, 1, 2, or 3; andrepresents a 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S;whereinindicates a point of attachment of the FAP-a binding ligand to the linker, L3, wherein the point of attachment can be through any of the carbon atoms of the 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle thereof;and stereoisomers and pharmaceutically acceptable salts thereof.

3. The compound of 2, wherein R4x is selected from H, -CH3, -CH2-phenyl, -CH(CH3)2, -CH2-OH, and -CH(OH)CH3.

4. The compound of claim 2, whereinis selected from:14244429.601_P18646-02wherein * indicates the point of attachment of the 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle to -(CH2)V-5. The compound of claim 2, wherein A comprises an FAP-a targeting moiety having the following structure:whereinindicates a point of attachment of the FAP-a binding ligand to the linker, L3, wherein the point of attachment can be through any of carbon atoms 5, 6, 7, or 8 of the quinolinyl ring thereof; and stereoisomers and pharmaceutically acceptable salts thereof.14344429.601_P18646-026. The compound of claim 5, wherein A is selected from:

7. The compound of claim 6, wherein A is selected from:and stereoisomers thereof.

8. The compound of claim 6, wherein A is selected from:

9. The compound of claim 1, wherein A comprises an FAP-a targeting moiety having the following structure:wherein:14444429.601_P18646-02Xi and X2 are each independently H or F; andR15x is selected from H, C1-6 alkyl, halogen, trihalomethoxyl, C1-6 alkoxyl, and 4- methoxyphenyl.

10. The compound of claim 1, wherein A comprises an FAP-a targeting moiety having the following structure:n NiH11. The compound of claim 1, wherein A comprises a targeting moiety for FAP-a having the following structure:

12. The compound of claim 1, wherein A comprises an FAP-a targeting moiety having the following structure:y(Rlx) (^2x)yy(R3x’)'HO^N5H—wherein:each y is independently an integer selected from 0, 1, and 2;R1x, R2x, and R3x', are each independently selected from H, OH, halogen, Ci-ealkyl, -O-Ci-ealkyl, and -S-Ci-ealkyl;14544429.601_P18646-02whereinindicates a point of attachment of the FAP-a binding ligand to the linker, L3, wherein the point of attachment can be through any of the carbon atoms of the 5 to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle thereof;and stereoisomers and pharmaceutically acceptable salts thereof.

13. The compound of claim 1, wherein P-X is selected from:wherein:each X is independently a radioisotope of a halogen;each n is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;each carbon of thenalkylene chain can be substituted with C1-C4 alkyl; and each R and R’ is independently H or C1-C4 alkyl.

14. The compound of claim 1, wherein P-X is selected from:14644429.601_P18646-0215. The compound of claim 1, wherein X is selected from18F,124I,1251,131I, and211At.

16. The compound of claim 1, wherein C comprises a chelating agent selected from DOTAGA (1,4,7, 10-tetraazacyclododececane,l-(glutaric acid)-4,7,10-triacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DOTASA (1,4,7,10-tetraazacyclododecane-1- (2- succinic acid)-4,7,10-triacetic acid), CB-DO2A (10-bis (carboxymethyl)- 1,4,7.10-tetraazabicyclo [5.5.2] tetradecane), DEPA (7- [2 - (B i s -carboxymethylamino)-ethyl] -4, 10-bis-carboxymethyl- 1,4,7,10-tetraaza-cyclododec- 1 -yl-acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-l-[4,7,10-tris(carboxymethyl)-1,4,7, 10-tetraazacyclododecan-l-yl]pentan-2-yl)amino]acetic acid)), TCMC (2-(4-isothiocyanotobenzyl)- 1,4,7, 10-tetraaza- 1,4,7, 10-tetra- (2-carbamonyl methyl)-cyclododecane), oxo-DO3A (l-oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid). p-NH2-Bn-Oxo-D03A (l-Oxa-4,7,10-tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A (( 1,8-A, A'-bis-(carboxymethyl)-l,4,8, 11-tetraazacyclotetradecane), MM-TE2A, DM-TE2A, CB-TE2A (4,ll-bis(carboxymethyl)-l,4,8,ll-14744429.601_P18646-02tetraazabicyclo[6.6.2]hexadecane), CB-TE1 A1P (4,8,11 -tetraazacyclotetradecane- 1 -(methanephosphonic acid)-8-(methanecarboxylic acid), CB-TE2P (1,4,8,11-tetraazacyclotetradecane-l,8-bis(methanephosphonic acid), TETA (1,4,8,11-tetraazacyclotetradecane- 1,4, 8, 11 -tetraacetic acid), NOTA (l,4,7-triazacyclononane-N, N', N"-triacetic acid), NODA ( 1,4, 7-triazacyclononane-l,4-di acetate ); NOD AG A (1,4,7-triazacyclononane.l -glutaric acid-4, 7-acetic acid), (NOTAGA) l,4,7-triazonane-1.4-diyl)diacetic acid DFO (Desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethl- [ 1,4, 7] triazonan-l-yl} -acetic acid), TACN-TM (N, N'. N", tris(2-mercaptoethyl)- 1,4,7-triazacyclononane), Diamsar (l,8-Diamino-3,6,10,13,16,19-hexaazabicyclo(6,6,6)eicosane, 3.6.10.13.16.19-Hexaazabicyclo[6.6.6]eicosane-l,8-diamine), Sarar (l-N-(4-aminobenzyl)-3, 6.10.13.16.19-hexaazabicyclo[6.6.6] eicosane- 1,8-diamine), AmBaSar (4-((8-amino- 3.6.10.13.16.19-hexaazabicyclo [6.6.6] icosane-l-ylamino) methyl) benzoic acid), macropa, and BaBaSar.

17. The compound of claim 1, wherein C comprises a chelating agent selected from:, CO2HL — N — \( CO2HCO2H14844429.601_P18646-0244429.601__P 18646-0244429.601_P18646-0244429.601_P18646-0218. The compound of claim 17, wherein the naturally occurring non-radioactive isotope of a metal is selected from Cu, Pb, Ac, Lu, Ga, Tb, Y, In, Re, Sm, Zr, Bi, Sc, Ho, Ra, Th, and Al.

19. The compound of claim 18, wherein the naturally occurring non-radioactive isotope of a metal is selected from27Al,45Sc,63Cu,65Cu,6yGa,7lGa,89Y,90Zr,91Zr,92Zr,94Zr,15244429.601_P18646-0296Zr,n3In,115In,144Sm,147Sm,148Sm,149Sm,150Sm,152Sm,154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th.

20. The compound of claim 1, wherein Laand Lb are each individually selected from (a), (b), (c). or (d):wherein:pi, p2, p3 and p4 may be in any order;ti and t2 are each an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;pi, p3. and p4 are each independently 0 or 1;P2 is an integer selected from 0, 1, 2, and 3, and when p2 is 2 or 3, each Ri is the same or different;mi, m2, m3, and ir are each an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8;Wi is selected from a bond, -S-, -C(=O)-, -C(=O)-NR-, and -NR-C(=O)-;W2is selected from a bond. -S-, -CH2-C(=O)-NR-, -C(=O)-, -NRC(=O)-. -NR'C(=O)NR-, -NRC(=S)NR'2-, -NRC(=O)O- -OC(=O)NR-, -OC(=O)-, -C(=O)NR-, -NR-C(=O)-, -C(=O)O-, - (O- CH2- CH2)q- and -(CH2-CH2-O)q, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;each R or R' is independently H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl;15344429.601_P18646-02Y can be present or absent and when present is selected from a nitrogen-containing heteroalkylene chain, a cyclic or bicylic heteroalkyl radical, and a triazole radical having thestructureeach Ri is independently H, Ci-Ce alkyl, C3-C12 aryl, -(CH2)q-C3-Ci2 aryl, -C4-C16 alkylaryl, or -(CH2)q-C4-Ci6 alkylaryl;R2 and R3 are each independently H, -(CH2)q-C3-Ci2 aryl, and -CO2R5, wherein R5 is selected from H, Ci-Ce alkyl, C3-C12 aryl, and C4-C16 alkylaryl, wherein R2 and R3 can be the same or different, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;V is selected from -C(O)-, -C(S)-, -NRC(O)-, -NRC(S)-, and -OC(O)-;pi, P2, P3 and p4 may be in any order;t2 is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;pi and p3 are each independently 0 or 1;P2 is an integer selected from 0, 1, 2, and 3, and when p2 is 2 or 3, each Ri is the same or different;mi and m2 are each an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8; W2is selected from a bond, -S-, -CH2-C(=O)-NR-, -C(=O)-, -NRC(=O)-, -NR'C(=O)NR-, -NRC(=S)NR'2-, -NRC(=O)O-, -OC(=O)NR-, -OC(=O)-, -C(=O)NR-, -NR-C(=O)-. -C(=O)O-, - (O- CH2- CH2)q- and -(CH2-CH2-O)q, wherein q is selected from 0, 1.2, 3, 4, 5, 6, 7, and 8;each R or R' is independently H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl;each Ri is independently H, Ci-Ce alkyl, C3-C12 aryl, -(CH2)q-C3-Ci2 aryl, -C4-C16 alkylaryl, or -(CH2)q-C4-Ci6 alkylaryl, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;15444429.601_P18646-02R2 and R3 are each independently H, -(CH2)q-C3-Ci2 aryl, and -CO2R5, wherein R5 is selected from H, Ci-Ce alkyl, C3-C12 aryl, and C4-C16 alkylaryl, wherein R2 and R3 can be the same or different, wherein q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; andV is selected from -C(O)-, -C(S)-, -NRC(O)-, -NRC(S)-, and -OC(O)-;(c) -Li-, -L2-L3-, or -L1-L2-L3-, wherein:Li is -NR-(CH2)q-[O-CH2-CH2-O]q-(CH2)q-C(=O)-;L2is -NR-(CH2)q-C(COOR5)-NR-; andL3 is -(O=)C-(CH2)q-C(=O)-;wherein each q is independently an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;R is selected from H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl; andR5 is selected from H, Ci-Ce alkyl, C3-C12 aryl, and C4-C16 alkylaryl;(d) -(CR6H)q-(CH2)q-C(=O)-NR-(CH2)q-O- or -NR-(CH2)q-O-; wherein:q is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8;Rs is H or -COORs;R is selected from H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl; andR5 is selected from H, Ci-Ce alkyl, C3-C12 aryl, and C4-C16 alkylaryl.

21. The compound of claim 20, wherein Y is selected from:(Rl6)z4hxix'; wherein Xi and X2 are each independently -CH- or N; each Ri6 is independently H or -C(=O)-ORi7, wherein R17 is C1-C4 alkyl; z4 is an integer selected from 0, 1, 2, 3, and 4;-N(Ri8)-(CH2)z5-N(Ri9)-; wherein Rig and R19 are each independently H or C1-C4 alkyl and z5 is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;15544429.601_P18646-0222. The compound of 20, wherein one or more of Laand Lb include one or more units selected from:15644429.601_P18646-02COOCH315744429.601_P18646-02wherein u is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;R is selected from H, alkyl, substituted alkyl, c ycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and -OR4, wherein R4 is selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, and substituted heterocycloalkyl; andR5 is selected from H, Ci-Ce alkyl, C3-C12 aryl, and C4-C16 alkylaryl.

23. The compound of claim 1, wherein the FAP-alpha binding moiety comprises:

24. The compound of claim 1, wherein P-X comprises:N25. The compound of claim 24, wherein P-X is 6-[18F]fluoronicotinamide:O15844429.601_P18646-0226. The compound of claim 1, wherein the chelator comprises 1,4,7,10-tetraazacyclododecane-l,4,7-tris-acetic acid-10 monoamide (DOTA-monoamide):

26. The compound of claim 1, wherein the linker. Lb. comprises a Ci-Cs alkylene chain.

27. The compound of claim 1, wherein the linker, La, comprises:

28. The compound of claim 1, wherein the La-(C)-Lb-P-X comprises:xo29. The compound of claim 1, wherein the compound of formula (I) is selected from:15944429.601_P18646-0244429.601_P 18646-0244429.601_P18646-0244429.601_P18646-0230. The compound of claim 29, wherein the compound of formula (I) is selected from:16344429.601_P18646-0244429.601_P18646-0244429.601_P18646-02wherein M is a naturally occurring non-radioactive isotope of a metal.

31. The compound of claim 30, wherein the compound of formula (I) is selected from:16644429.601_P18646-0244429.601_P18646-0244429.601_P18646-0244429.601_P18646-0218Fo; ana o wherein M is a naturally occurring non-radioactive isotope of a metal.

32. The compound of claim 1, wherein the compound of formula (I) comprises:owherein M is selected from113In / 115In,45Sc, and69Ga / 71Ga.

33. The compound of claim 32, wherein the compound of formula (I) comprises:wherein M is71Ga / 71Ga.

34. A pharmaceutical composition comprising the compound of any of claims 1-33.

35. The composition of claim 34, further comprising one or more of pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

36. A method for imaging a disease or disorder associated with fibroblast-activation protein-a (FAP-a), the method comprising administering a compound according to any of claims 1-33, or a pharmaceutical composition thereof, and obtaining an image.17044429.601_P18646-0237. A method for inhibiting fibroblast-activation protein-a (FAP-a), the method comprising administering to a subject in need thereof an effective amount of a compound according to any of claims 1-33, or a pharmaceutical composition thereof.

38. The method of claim 36, wherein the (FAP-a)-related disease or disorder is selected from a proliferative disease, a disease characterized by tissue remodeling and / or chronic inflammation, a disorder involving endocrinological dysfunction, and a blood clotting disorder.

39. The method of claim 38, wherein the proliferative disease is selected from breast cancer, colorectal cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, melanoma, fibrosarcoma, bone and connective tissue sarcomas, renal cell carcinoma, giant cell carcinoma, squamous cell carcinoma, gastric cancer, a glioma, and adenocarcinoma, including pancreatic ductal adenocarcinoma.

40. A method for synthesizing:the method comprising:(a) providing a trimethylammonium precursor compound (3):(b) contacting the trialkylammonium precursor compound (3) with18F / 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane in the presence of a base in a polar, aprotic solvent at a first tempearture to form [18F]1:17144429.601_P18646-02([18F]1); and(c) contacting ([18F] 1) with M3+in a buffer at a second temperature to form:wherein M is a naturally occurring non-radioactive isotope of a metal.

41. The method of claim 40, wherein M is selected from Cu, Pb, Ac, Lu, Ga, Tb, Y, In, Re, Sm, Zr, Bi, Sc, Ho, Ra, Th, and Al.

42. The method of claim 41, wherein M is selected from27Al,45Sc,63Cu,65Cu,69Ga,71Ga,89Y.90Zr.91Zr,92Zr,94Zr,96Zr,113In,115In,144Sm,147Sm,148Sm,149Sm,150Sm.152Sm.154Sm,159Tb,175Lu,185Re,187Re,204Pb,206Pb,207Pb,208Pb,209Bi,227Ac, and232Th.

43. The method of claim 42, wherein M is selected from113In / 115In,45Sc, and69Ga / 7lGa.

44. The method of claim 40, wherein M3+is a salt selected from113In / 115InCl3,45SCC13,113In / 115In(NO3)3,45Sc(NO3)3, and69Ga / 71Ga(NO3)3.

45. The method of claim 40, wherein the base is potassium oxalate.

46. The method of claim 40, wherein the polar, aprotic solvent comprises dimethylsulfoxide (DMSO).

47. The method of claim 40, wherein the first temperature is about 90 °C.17244429.601_P18646-0248. The method of claim 40, wherein the buffer is an acetate buffer.

49. The method of claim 40, wherein step (c) is conducted at a pH of about 5.

50. The method of claim 40, wherein the second temperature is about 70 °C.

51. The method of claim 40, wherein [18F]-M-1 is purified by preparative high performance liquid chromatography.

52. The method of claim 51, comprising further purifying [18F]-M-1 by solid-phase extraction (SPE).

53. The method of claim 40, wherein [18F]-M-1 has a radiochemical yield of about 25%.

54. The method of claim 40, wherein [18F]-M-1 has a radiochemical purity greater than about 95%.

55. The method of claim 40, wherein [18F]-M-1 has a molar (specific) radioactivity between about 2000 to about 5000 Ci / mmol.17344429.601_P18646-02

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